Pdx1-expressing dorsal and ventral foregut endoderm

ABSTRACT

Disclosed herein are cell cultures comprising dorsal and/or ventral PDX1-positive foregut endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified dorsal and/or ventral PDX1-positive foregut endoderm cells as well as methods for enriching, isolating and purifying dorsal and/or ventral PDX1-positive foregut endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of dorsal and/or ventral PDX1-positive foregut endoderm cells, are also disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/588,693, filed Oct. 27, 2006, entitled “PDX1-EXPRESSING DORSAL ANDVENTRAL FOREGUT ENDODERM,” which is a nonprovisional application of andclaims priority to U.S. Provisional Patent Application No. 60/730,917,entitled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM, filed Oct.27, 2005, the disclosures of which are incorporated herein by referencein their entireties.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and cellbiology. In particular, the present invention relates to compositionscomprising mammalian foregut endoderm cells and compositions comprisingdorsal and/or ventral PDX1-positive foregut endoderm cells and methodsof making, isolating and using such cells.

BACKGROUND

Human pluripotent stem cells, such as embryonic stem (ES) cells andembryonic germ (EG) cells, were first isolated in culture withoutfibroblast feeders in 1994 (Bongso et al., 1994) and with fibroblastfeeders (Hogan, 1997). Later, Thomson, Reubinoff and Shamblottestablished continuous cultures of human ES and EG cells usingmitotically inactivated mouse feeder layers (Reubinoff et al., 2000;Shamblott et al., 1998; Thomson et al., 1998).

Human ES and EG cells (hESCs) offer unique opportunities forinvestigating early stages of human development as well as fortherapeutic intervention in several disease states, such as diabetesmellitus and Parkinson's disease. For example, the use ofinsulin-producing β-cells derived from hESCs would offer a vastimprovement over current cell therapy procedures that utilize cells fromdonor pancreases for the treatment of diabetes. However, presently it isnot known how to generate an insulin-producing β-cell from hESCs. Assuch, current cell therapy treatments for diabetes mellitus, whichutilize islet cells from donor pancreases, are limited by the scarcityof high quality islet cells needed for transplant. Cell therapy for asingle Type I diabetic patient requires a transplant of approximately8×10⁸ pancreatic islet cells. (Shapiro et al., 2000; Shapiro et al.,2001a; Shapiro et al., 2001b). As such, at least two healthy donororgans are required to obtain sufficient islet cells for a successfultransplant. Human embryonic stem cells offer a source of startingmaterial from which to develop substantial quantities of high qualitydifferentiated cells for human cell therapies.

Two properties that make hESCs uniquely suited to cell therapyapplications are pluripotence and the ability to maintain these cells inculture for prolonged periods. Pluripotency is defined by the ability ofhESCs to differentiate to derivatives of all 3 primary germ layers(endoderm, mesoderm, ectoderm) which, in turn, form all somatic celltypes of the mature organism in addition to extraembryonic tissues (e.g.placenta) and germ cells. Although pluripotency imparts extraordinaryutility upon hESCs, this property also poses unique challenges for thestudy and manipulation of these cells and their derivatives. Owing tothe large variety of cell types that may arise in differentiating hESCcultures, the vast majority of cell types are produced at very lowefficiencies. Additionally, success in evaluating production of anygiven cell type depends critically on defining appropriate markers.Achieving efficient, directed differentiation is of great importance fortherapeutic application of hESCs.

In order to use hESCs as a starting material to generate cells that areuseful in cell therapy applications, it would be advantageous toovercome the foregoing problems. For example, in order to achieve thelevel of cellular material required for islet cell transplantationtherapy, it would be advantageous to efficiently direct hESCs toward thepancreatic islet/β-cell lineage at the very earliest stages ofdifferentiation.

In addition to efficient direction of the differentiation process, itwould also be beneficial to isolate and characterize intermediate celltypes along the differentiation pathway towards the pancreaticislet/β-cell lineage and to use such cells as appropriate lineageprecursors for further steps in the differentiation.

SUMMARY OF THE INVENTION

Embodiments of the present invention relate to cell cultures ofPDX1-negative foregut endoderm cells (foregut endoderm cells). In someembodiments, the foregut endoderm expresses the HNF1b and FOXA1 markersbut does not substantially express PDX1. Other embodiments of thepresent invention relate to cell cultures of PDX1-positive,dorsally-biased, foregut endoderm cells (dorsal PDX1-positive foregutendoderm cells). In some embodiments, the PDX1-positive,dorsally-biased, foregut endoderm cells express one or more markersselected from Table 3 and/or one or more markers selected from Table 4.Additional embodiments, relate to cell cultures of PDX1-positive,ventrally-biased, foregut endoderm cells (ventral PDX1-positive foregutendoderm cells). In some embodiments, the PDX1-positive,ventrally-biased, foregut endoderm cells express one or more markersselected from Table 3 but do not substantially express a marker selectedfrom Table 4 as compared to the expression of the same marker inPDX1-positive, dorsally-biased, foregut endoderm cells.

Additional embodiments of the present invention relate to enriched,isolated and/or purified cell populations comprising PDX1-negativeforegut endoderm cells. Other embodiments relate to PDX1-positive,dorsally-biased, foregut endoderm cells. Still other embodiments relateto enriched, isolated and/or purified cell populations comprisingPDX1-positive, ventrally-biased, foregut endoderm cells.

Aspects of the present invention also relate to methods or processes forthe production of cell cultures of PDX1-negative foregut endoderm cellsfrom definitive endoderm cells. Such processes include reducing oreliminating TGFβ superfamily growth factor signaling in a cell cultureor cell population of definitive endoderm cells. In some embodiments,reducing or eliminating TGFβ superfamily growth factor signaling ismediated by diluting or removing an exogenously added TGFβ superfamilygrowth factor, such as activin A, from the cell culture or cellpopulation of definitive endoderm. In some embodiments, differentiationof definitive endoderm cells to foregut endoderm cells is enhanced byproviding the definitive endoderm cell culture or cell population withan FGF-family growth factor and/or a hedgehog pathway inhibitor. In someembodiments, the definitive endoderm cells are derived from stem cells.Preferably, the stem cells are embryonic stem cells. Even morepreferably, the stem cells are human embryonic stem cells (hESCs). Insome embodiments, the PDX1-negative foregut endoderm cells aredifferentiated to PDX1-positive endoderm cells (pancreatic endodermcells) by the addition of a retinoid, such as retinoic acid. Otheraspects relate to methods or processes for the production of cellcultures of PDX1-positive, dorsally-biased, foregut endoderm cells. Suchprocesses include providing definitive endoderm cells with retinoicacid. In some embodiments, the definitive endoderm cells are derivedfrom stem cells. Preferably, the stem cells are embryonic stem cells.Even more preferably, the stem cells are human embryonic stem cells(hESCs). Further aspects of the present invention relate to methods orprocesses for the production of cell cultures of PDX1-positive,ventrally-biased, foregut endoderm cells. Such processes includeproviding definitive endoderm cells with an FGF-family growth factor. Insome embodiments, the definitive endoderm cells are derived from stemcells. Preferably, the stem cells are embryonic stem cells. Even morepreferably, the stem cells are hESCs.

Additional embodiments of the present invention relate to methods ofenriching, isolating and/or purifying PDX1-negative foregut endodermcells. In such embodiments, PDX1-negative foregut endoderm cells areseparated from other cells in the cell population by using an antibody,ligand or other molecule that binds to a molecule that is expressed onthe cell surface of PDX1-negative foregut endoderm cells, such as a cellsurface molecule. Other embodiments of the present invention relate tomethods of enriching, isolating and/or purifying PDX1-positive,dorsally-biased, foregut endoderm cells. In such embodiments,PDX1-positive, dorsally-biased, foregut endoderm cells are separatedfrom other cells in the cell population by using an antibody, ligand orother molecule that binds to a molecule that is expressed on the cellsurface of PDX1-positive, dorsally-biased, foregut endoderm cells, suchas a cell surface molecule selected from Table 3 or a cell surfacemolecule selected from Table 4. Still other embodiments of the presentinvention relate to methods of enriching, isolating and/or purifyingPDX1-positive, ventrally-biased, foregut endoderm cells. In suchembodiments, PDX1-positive, ventrally-biased, foregut endoderm cells areseparated from other cells in the cell population by using an antibody,ligand or other molecule that binds to a molecule that is expressed onthe cell surface of PDX1-positive, ventrally-biased, foregut endodermcells, such as a cell surface molecule selected from Table 3.

Embodiments of the present invention relate to additional methods ofenriching, isolating and/or purifying PDX1-negative foregut endodermcells. In such embodiments, pluripotent or multipotent cells that areprecursors to PDX1-negative foregut endoderm cells are engineered tocontain a fluorescent reporter gene under control of a promoter thatendogenously controls the expression of a marker gene such as HNF1b orFOXA1. The fluorescently-tagged PDX1-negative foregut endoderm cells arethen separated from other cells in the cell population by fluorescenceactivated cell sorting (FACS). Still other embodiments of the presentinvention relate to additional methods of enriching, isolating and/orpurifying PDX1-positive, dorsally-biased, foregut endoderm cells. Insuch embodiments, pluripotent or multipotent PDX1-negative cells thatare precursors to PDX1-positive cells are engineered to contain afluorescent reporter gene under control of a promoter that endogenouslycontrols the expression of a marker gene selected from Table 3 or Table4. The fluorescently-tagged PDX1-positive, dorsally-biased, foregutendoderm cells are then separated from other cells in the cellpopulation by fluorescence activated cell sorting (FACS). Yet otherembodiments of the present invention relate to additional methods ofenriching, isolating and/or purifying PDX1-positive, ventrally-biased,foregut endoderm cells. In such embodiments, pluripotent or multipotentPDX1-negative cells that are precursors to PDX1-positive cells areengineered to contain a fluorescent reporter gene under control of apromoter that endogenously controls the expression of a marker geneselected from Table 3. The fluorescently-tagged PDX1-positive,ventrally-biased, foregut endoderm cells are then separated from othercells in the cell population by FACS.

Further embodiments of the present invention relate to methods ofidentifying a differentiation factor capable of promoting thedifferentiation of human PDX1-negative foregut endoderm cells in a cellpopulation comprising human cells. The method includes the steps ofobtaining a cell population comprising human PDX1-negative foregutendoderm cells, providing a candidate differentiation factor to the cellpopulation, determining expression of a marker, such as HNF1b, FOXA1 orPDX1, in the cell population at a first time point and determiningexpression of the same marker in the cell population at a second timepoint. In such embodiments, the second time point is subsequent to thefirst time point and the second time point is subsequent to providingthe cell population with the candidate differentiation factor. Ifexpression of the marker in the cell population at the second time pointis increased or decreased as compared to the expression of the marker inthe cell population at the first time point, then the candidatedifferentiation factor is capable of promoting the differentiation ofthe human PDX1-negative foregut endoderm cells. Still furtherembodiments of the present invention relate to methods of identifying adifferentiation factor capable of promoting the differentiation of humanPDX1-positive, dorsally-biased, foregut endoderm cells in a cellpopulation comprising human cells. The method includes the steps ofobtaining a cell population comprising human PDX1-positive,dorsally-biased, foregut endoderm cells, providing a candidatedifferentiation factor to the cell population, determining expression ofa marker, such as a marker selected from Table 3 or a marker selectedfrom Table 4, in the cell population at a first time point anddetermining expression of the same marker in the cell population at asecond time point. In such embodiments, the second time point issubsequent to the first time point and the second time point issubsequent to providing the cell population with the candidatedifferentiation factor. If expression of the marker in the cellpopulation at the second time point is increased or decreased ascompared to the expression of the marker in the cell population at thefirst time point, then the candidate differentiation factor is capableof promoting the differentiation of the human PDX1-positive,dorsally-biased, foregut endoderm cells. Yet further embodiments of thepresent invention relate to methods of identifying a differentiationfactor capable of promoting the differentiation of human PDX1-positive,ventrally-biased, foregut endoderm cells in a cell population comprisinghuman cells. The method includes the steps of obtaining a cellpopulation comprising human PDX1-positive, ventrally-biased, foregutendoderm cells, providing a candidate differentiation factor to the cellpopulation, determining expression of a marker, such as a markerselected from Table 3, in the cell population at a first time point anddetermining expression of the same marker in the cell population at asecond time point. In such embodiments, the second time point issubsequent to the first time point and the second time point issubsequent to providing the cell population with the candidatedifferentiation factor. If expression of the marker in the cellpopulation at the second time point is increased or decreased ascompared to the expression of the marker in the cell population at thefirst time point, then the candidate differentiation factor is capableof promoting the differentiation of the human PDX1-positive,ventrally-biased, foregut endoderm cells.

In certain jurisdictions, there may not be any generally accepteddefinition of the term “comprising.” As used herein, the term“comprising” is intended to represent “open” language which permits theinclusion of any additional elements. With this in mind, additionalembodiments of the present inventions are described with reference tothe numbered paragraphs below:

1. A cell culture comprising human cells wherein at least about 26% ofsaid human cells are pancreatic-duodenal homoebox factor-1 (PDX1)positive, dorsally-biased, foregut endoderm cells that express at leastone marker selected from Table 3, said PDX1-positive, dorsally-biased,foregut endoderm cells being multipotent cells that can differentiateinto cells of the dorsal pancreatic bud.

2. The cell culture of paragraph 1, wherein said marker selected fromTable 3 is a marker expressed on the cell surface.

3. The cell culture of paragraph 2, wherein said marker expressed on thecell surface is selected from the group consisting of CDH6, GABRA2,GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

4. The cell culture of paragraph 1, wherein said PDX1-positive,dorsally-biased, foregut endoderm cells express at least one markerselected from Table 4.

5. The cell culture of paragraph 4, wherein said marker selected fromTable 4 is a marker expressed on the cell surface.

6. The cell culture of paragraph 5, wherein said marker expressed on thecell surface is selected from the group consisting of ADORA2A, CD47,EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4and XPR1.

7. The cell culture of paragraph 1, wherein at least about 30% of saidhuman cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

8. The cell culture of paragraph 1, wherein at least about 40% of saidhuman cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

9. The cell culture of paragraph 1, wherein at least about 50% of saidhuman cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

10. The cell culture of paragraph 1, wherein at least about 60% of saidhuman cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

11. The cell culture of paragraph 1, wherein at least about 75% of saidhuman cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

12. The cell culture of paragraph 1, wherein human feeder cells arepresent in said culture, and wherein at least about 2% of human cellsother than said human feeder cells are PDX1-positive, dorsally biased,foregut endoderm cells.

13. The cell culture of paragraph 1, wherein the expression of PDX1 isgreater than the expression of a marker selected from the groupconsisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in saidPDX1-positive, dorsally biased, foregut endoderm cells.

14. The cell culture of paragraph 1, wherein said cell culture issubstantially free of cells selected from the group consisting ofvisceral endodermal cells, parietal endodermal cells and neural cells.

15. The cell culture of paragraph 1 further comprising a retinoid.

16. The cell culture of paragraph 15, wherein said retinoid is retinoicacid (RA).

17. The cell culture of paragraph 16 further comprising B27.

18. A cell culture comprising human cells wherein at least about 2% ofsaid human cells are pancreatic-duodenal homoebox factor-1 (PDX1)positive, ventrally-biased, foregut endoderm cells that express at leastone marker selected from Table 3, said PDX1-positive, ventrally-biased,foregut endoderm cells being multipotent cells that can differentiateinto cells of the ventral pancreatic bud.

19. The cell culture of paragraph 18, wherein said marker selected fromTable 3 is a marker expressed on the cell surface.

20. The cell culture of paragraph 19, wherein said marker expressed onthe cell surface is selected from the group consisting of CDH6, GABRA2,GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

21. The cell culture of paragraph 18, wherein said PDX1-positive,ventrally-biased, foregut endoderm cells do not substantially expressone or more markers selected from Table 4.

22. The cell culture of paragraph 18, wherein at least about 5% of saidhuman cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

23. The cell culture of paragraph 18, wherein at least about 10% of saidhuman cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

24. The cell culture of paragraph 18, wherein at least about 25% of saidhuman cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

25. The cell culture of paragraph 18, wherein at least about 50% of saidhuman cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

26. The cell culture of paragraph 18, wherein at least about 75% of saidhuman cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

27. The cell culture of paragraph 18, wherein human feeder cells arepresent in said culture, and wherein at least about 2% of human cellsother than said human feeder cells are PDX1-positive, ventrally-biased,foregut endoderm cells.

28. The cell culture of paragraph 18, wherein the expression of PDX1 isgreater than the expression of a marker selected from the groupconsisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in saidPDX1-positive, ventrally-biased, foregut endoderm cells.

29. The cell culture of paragraph 18, wherein said cell culture issubstantially free of cells selected from the group consisting ofvisceral endodermal cells, parietal endodermal cells and neural cells.

30. The cell culture of paragraph 18 further comprising a retinoid.

31. The cell culture of paragraph 30, wherein said retinoid is retinoicacid (RA).

32. The cell culture of paragraph 31 further comprising B27.

33. A cell population comprising cells wherein at least about 90% ofsaid cells are human PDX1-positive, dorsally-biased, foregut endodermcells that express at least one marker selected from Table 3, saidPDX1-positive, dorsally-biased, foregut endoderm cells being multipotentcells that can differentiate into cells of the dorsal pancreatic bud.

34. The cell population of paragraph 33, wherein said marker selectedfrom Table 3 is a marker expressed on the cell surface.

35. The cell population of paragraph 34, wherein said marker expressedon the cell surface is selected from the group consisting of CDH6,GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

36. The cell population of paragraph 33, wherein said PDX1-positive,dorsally-biased, foregut endoderm cells express at least one markerselected from Table 4.

37. The cell population of paragraph 36, wherein said marker selectedfrom Table 4 is a marker expressed on the cell surface.

38. The cell population of paragraph 37, wherein said marker expressedon the cell surface is selected from the group consisting of ADORA2A,CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3, SLC30A4, SLICK,SLITRK4 and XPR1.

39. The cell population of paragraph 33, wherein at least about 95% ofsaid cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

40. The cell population of paragraph 33, wherein at least about 98% ofsaid cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

41. The cell population of paragraph 33, wherein the expression of PDX1is greater than the expression of a marker selected from the groupconsisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positive,dorsally-biased, foregut endoderm cells.

42. A cell population comprising cells wherein at least about 90% ofsaid cells are human PDX1-positive, ventrally-biased, foregut endodermcells that express at least one marker selected from Table 3, saidPDX1-positive, ventrally-biased, foregut endoderm cells beingmultipotent cells that can differentiate into cells of the ventralpancreatic bud.

43. The cell population of paragraph 42, wherein said marker selectedfrom Table 3 is a marker expressed on the cell surface.

44. The cell population of paragraph 43, wherein said marker expressedon the cell surface is selected from the group consisting of CDH6,GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

45. The cell population of paragraph 42, wherein said PDX1-positive,ventrally-biased, foregut endoderm cells do not substantially expressone or more markers selected from Table 4.

46. The cell population of paragraph 42, wherein at least about 95% ofsaid cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

47. The cell population of paragraph 42, wherein at least about 98% ofsaid cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

48. The cell population of paragraph 42, wherein the expression of PDX1is greater than the expression of a marker selected from the groupconsisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positive,ventrally-biased, foregut endoderm cells.

49. A method of producing PDX1-positive, dorsally-biased, foregutendoderm cells, said method comprising the steps of obtaining a cellpopulation comprising PDX1-negative definitive endoderm cells andproviding said cell population with a retinoid in an amount sufficientto promote differentiation of at least 26% of said PDX1-negativedefinitive endoderm cell population to PDX1-positive, dorsally-biased,foregut endoderm cells that express at least one marker selected fromTable 3, wherein said PDX1-positive, dorsally-biased, foregut endodermcells are multipotent cells that can differentiate into cells of thedorsal pancreatic bud.

50. The method of paragraph 49, wherein said PDX1-positive,dorsally-biased, foregut endoderm cells also express at least one markerselected from Table 4.

51. The method of paragraph 50 further comprising the step of allowingsufficient time for PDX1-positive, dorsally-biased, foregut endodermcells to form, wherein said sufficient time for PDX1-positive,dorsally-biased, foregut endoderm cells to form has been determined bydetecting the presence of a marker from Table 4 in dorsally-biasedforegut endoderm cells in said cell population.

52. The method of paragraph 50, wherein the expression of said markerselected from Table 3 or Table 4 is determined by quantitativepolymerase chain reaction (Q-PCR).

53. The method of paragraph 50, wherein the expression of said markerselected from Table 3 or Table 4 is determined by immunocytochemistry.

54. The method of paragraph 49, wherein the expression of PDX1 isgreater than the expression of a marker selected from the groupconsisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in saidPDX1-positive, dorsally-biased, foregut endoderm cells.

55. The method of paragraph 49, wherein said retinoid is RA.

56. The method of paragraph 55, wherein RA is provided in aconcentration ranging from about 0.5 μM to about 50 μM.

57. The method of paragraph 56, wherein RA is provided in aconcentration ranging from about 1 μM to about 20 μM.

58. The method of paragraph 57, wherein RA is provided in aconcentration of about 2 μM.

59. The method of paragraph 55, wherein RA is provided when said cultureis about 5-days-old.

60. The method of paragraph 49 further comprising providing B27 to saidculture.

61. The method of paragraph 60, wherein said B27 is provided in aconcentration ranging from about 0.1% to about 20% of the total medium.

62. The method of paragraph 61, wherein B27 is provided in aconcentration ranging from about 0.5% to about 2% of the total medium.

63. The method of paragraph 62, wherein B27 is provided in aconcentration of about 0.5% of the total medium.

64. The method of paragraph 60, wherein B27 is provided at approximatelythe same time as said retinoid.

65. The method of paragraph 49 further comprising providing activin A tosaid culture.

66. The method of paragraph 65, wherein activin A is provided in aconcentration ranging from about 10 ng/ml to about 200 ng/ml.

67. The method of paragraph 66, wherein activin A is provided in aconcentration ranging from about 20 ng/ml to about 100 ng/ml.

68. The method of paragraph 67, wherein activin A is provided in aconcentration of about 25 ng/ml.

69. The method of paragraph 49, wherein said PDX1-positive,dorsally-biased, foregut endoderm cells are grown in CMRL medium.

70. The method of paragraph 69, wherein said CMRL medium comprises RA atabout 2 μM, activin A at about 25 ng/ml and B27 at about 0.5% of thetotal medium.

71. The method of paragraph 49, wherein said step of obtaining a cellpopulation comprising PDX1-negative definitive endoderm cells comprisesobtaining a cell population comprising pluripotent human cells,providing said cell population with at least one growth factor of theTGFβ superfamily in an amount sufficient to promote differentiation ofsaid pluripotent cells to definitive endoderm cells and allowingsufficient time for definitive endoderm cells to form, wherein saidsufficient time for definitive endoderm cells to form has beendetermined by detecting the presence of definitive endoderm cells insaid cell population.

72. A PDX1-positive, dorsally-biased, foregut endoderm cell produced bythe method of paragraph 49.

73. A method of producing PDX1-positive, ventrally-biased, foregutendoderm cells, said method comprising the steps of obtaining a cellpopulation comprising PDX1-negative definitive endoderm cells andproviding said cell population with an FGF-family growth factor in anamount sufficient to promote differentiation of at least a portion ofsaid PDX1-negative definitive endoderm cell population to PDX1-positive,ventrally-biased, foregut endoderm cells that express at least onemarker selected from Table 3, wherein said PDX1-positive,ventrally-biased, foregut endoderm cells are multipotent cells that candifferentiate into cells of the ventral pancreatic bud.

74. The method of paragraph 73, wherein said cell population isdifferentiated in the absence of RA.

75. The method of paragraph 73, wherein said PDX1-positive,ventrally-biased, foregut endoderm cells do not express one or moremarkers selected from Table 4.

76. The method of paragraph 75 further comprising the step of allowingsufficient time for PDX1-positive, ventrally-biased, foregut endodermcells to form, wherein said sufficient time for PDX1-positive,ventrally-biased, foregut endoderm cells to form has been determined bydetecting the presence of a marker from Table 3 in ventrally-biasedforegut endoderm cells in said cell population.

77. The method of paragraph 76, wherein the expression of said markerselected from Table 3 is determined by quantitative polymerase chainreaction (Q-PCR).

78. The method of paragraph 76, wherein the expression of said markerselected from Table 3 is determined by immunocytochemistry.

79. The method of paragraph 73, wherein the expression of PDX1 isgreater than the expression of a marker selected from the groupconsisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in saidPDX1-positive, ventrally-biased, foregut endoderm cells.

80. The method of paragraph 73, wherein said FGF-family growth factor isFGF-10, said FGF-10 being provided in a concentration ranging from about5 ng/ml to about 500 ng/ml.

81. The method of paragraph 80, wherein FGF-10 is provided in aconcentration ranging from about 10 ng/ml to about 100 ng/ml.

82. The method of paragraph 81, wherein FGF-10 is provided in aconcentration of about 50 ng/ml.

83. The method of paragraph 73, wherein said FGF-family growth factor isprovided when said culture is about 3-days-old.

84. The method of paragraph 73 further comprising providing B27 to saidculture.

85. The method of paragraph 84, wherein said B27 is provided in aconcentration ranging from about 0.1% to about 20% of the total medium.

86. The method of paragraph 85, wherein B27 is provided in aconcentration ranging from about 0.5% to about 2% of the total medium.

87. The method of paragraph 86, wherein B27 is provided in aconcentration of about 0.5% of the total medium.

88. The method of paragraph 84, wherein B27 is provided at approximatelythe same time as said FGF-family growth factor.

89. The method of paragraph 73 further comprising providing a hedgehoginhibitor to said culture

90. The method of paragraph 89, wherein said hedgehog inhibitor isKAAD-cyclopamine, said KAAD-cyclopamine being provided in aconcentration of about 0.1 μM to about 50 μM.

91. The method of paragraph 90, wherein KAAD-cyclopamine is provided ina concentration ranging from about 0.5 μM to about 10 μM.

92. The method of paragraph 91, wherein KAAD-cyclopamine is provided ina concentration of about 0.5 μM.

93. The method of paragraph 73, wherein said PDX1-positive,ventrally-biased, foregut endoderm cells are grown in CMRL medium.

94. The method of paragraph 93, wherein said CMRL medium comprisesFGF-10 at about 50 ng/ml, KAAD-cyclopamine at about 0.5 μM, and B27 atabout 0.5% of the total medium.

95. The method of paragraph 94, wherein said CMRL medium lacks RA.

96. The method of paragraph 73, wherein said step of obtaining a cellpopulation comprising PDX1-negative definitive endoderm cells comprisesobtaining a cell population comprising pluripotent human cells,providing said cell population with at least one growth factor of theTGFβ superfamily in an amount sufficient to promote differentiation ofsaid pluripotent cells to definitive endoderm cells and allowingsufficient time for definitive endoderm cells to form, wherein saidsufficient time for definitive endoderm cells to form has beendetermined by detecting the presence of definitive endoderm cells insaid cell population.

97. A PDX1-positive, ventrally-biased, foregut endoderm cell produced bythe method of paragraph 73.

98. A method of producing a cell population enriched in PDX1-positive,dorsally-biased, foregut endoderm cells, said method comprising thesteps of differentiating cells in a population of PDX1-negativedefinitive endoderm cells so as to produce PDX1-positive,dorsally-biased, foregut endoderm cells, said PDX1-positive,dorsally-biased, foregut endoderm cells being multipotent cells that candifferentiate into cells of the dorsal pancreatic bud, providing to saidcell population a reagent which binds to a marker expressed in saidPDX1-positive, dorsally-biased, foregut endoderm cells but which is notsubstantially expressed in other cell types present in said cellpopulation and separating said PDX1-positive, dorsally-biased, foregutendoderm cells bound to said reagent from said other cell types presentin said cell population, thereby producing a cell population enriched inPDX1-positive, dorsally-biased, foregut endoderm cells.

99. The method for paragraph 98, wherein said marker is selected fromthe group consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10,SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4 and XPR1.

100. The method for paragraph 98, wherein said marker is selected fromthe group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3,LGALS3/GALIG, SERPINF2 and SLC27A2.

101. The method of paragraph 98, wherein the differentiating stepfurther comprises obtaining a cell population comprising PDX1-negativedefinitive endoderm cells, providing said cell population with aretinoid in an amount sufficient to promote differentiation of saidPDX1-negative definitive endoderm cells to PDX1-positive,dorsally-biased, foregut endoderm cells, said PDX1-positive,dorsally-biased, foregut endoderm cells being multipotent cells that candifferentiate into cells of the dorsal pancreatic bud, and allowingsufficient time for PDX1-positive, dorsally-biased, foregut endodermcells to form, wherein said sufficient time for PDX1-positive,dorsally-biased, foregut endoderm cells to form has been determined bydetecting the presence of PDX1-positive, dorsally-biased, foregutendoderm cells in said cell population.

102. The method of paragraph 101, wherein the providing step furthercomprises providing B27.

103. The method of paragraph 101, wherein detecting comprises detectingthe expression of at least one marker selected from Table 4.

104. The method of paragraph 98, wherein at least about 95% of saidcells are PDX1-positive, dorsally-biased, foregut endoderm cells.

105. The method of paragraph 98, wherein at least about 98% of saidcells are PDX1-positive, dorsally-biased, foregut endoderm cells.

106. The method of paragraph 98, wherein said reagent is an antibody

107. The method of paragraph 106, wherein said antibody has affinity fora cell surface polypeptide selected from the group consisting ofADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3, SLC30A4,SLICK, SLITRK4, XPR1, CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3,LGALS3/GALIG, SERPINF2 and SLC27A2.

108. An enriched population of PDX1-positive, dorsally-biased, foregutendoderm cells produced by the method of paragraph 98.

109. A method of producing a cell population enriched in PDX1-positive,ventrally-biased, foregut endoderm cells, said method comprising thesteps of differentiating cells in a population of PDX1-negativedefinitive endoderm cells so as to produce PDX1-positive,ventrally-biased, foregut endoderm cells, said PDX1-positive,ventrally-biased, foregut endoderm cells being multipotent cells thatcan differentiate into cells of the ventral pancreatic bud, providing tosaid cell population a reagent which binds to a marker expressed in saidPDX1-positive, ventrally-biased, foregut endoderm cells but which is notsubstantially expressed in other cell types present in said cellpopulation and separating said PDX1-positive, ventrally-biased, foregutendoderm cells bound to said reagent from said other cell types presentin said cell population, thereby producing a cell population enriched inPDX1-positive, ventrally-biased, foregut endoderm cells.

110. The method for paragraph 109, wherein said marker is selected fromthe group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3,LGALS3/GALIG, SERPINF2 and SLC27A2.

111. The method of paragraph 109, wherein the differentiating stepfurther comprises obtaining a cell population comprising PDX1-negativedefinitive endoderm cells, providing said cell population with anFGF-family growth factor in an amount sufficient to promotedifferentiation of said PDX1-negative definitive endoderm cells toPDX1-positive, ventrally-biased, foregut endoderm cells, saidPDX1-positive, ventrally-biased, foregut endoderm cells beingmultipotent cells that can differentiate into cells of the ventralpancreatic bud, and allowing sufficient time for PDX1-positive,ventrally-biased, foregut endoderm cells to form, wherein saidsufficient time for PDX1-positive, ventrally-biased, foregut endodermcells to form has been determined by detecting the presence ofPDX1-positive, ventrally-biased, foregut endoderm cells in said cellpopulation.

112. The method of paragraph 111, wherein the providing step furthercomprises providing B27.

113. The method of paragraph 111, wherein detecting comprises detectingthe expression of at least one marker selected from Table 3.

114. The method of paragraph 109, wherein at least about 95% of saidcells are PDX1-positive, ventrally-biased, foregut endoderm cells.

115. The method of paragraph 109, wherein at least about 98% of saidcells are PDX1-positive, ventrally-biased, foregut endoderm cells.

116. The method of paragraph 109, wherein said reagent is an antibody

117. The method of paragraph 116, wherein said antibody has affinity fora cell surface polypeptide selected from the group consisting of CDH6,GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

118. An enriched population of PDX1-positive, ventrally-biased, foregutendoderm cells produced by the method of paragraph 109.

119. A method of producing a cell population enriched in PDX1-positive,dorsally-biased, foregut endoderm cells, said method comprising thesteps of obtaining a population of pluripotent cells, wherein at leastone cell of said pluripotent cell population comprises at least one copyof a nucleic acid under the control of the a promoter of any one of themarker genes selected from Table 4, said nucleic acid comprising asequence encoding a fluorescent protein or a biologically activefragment thereof, differentiating said pluripotent cells so as toproduce PDX1-positive, dorsally-biased foregut endoderm cells, saidPDX1-positive, dorsally-biased, foregut endoderm cells being multipotentcells that can differentiate into cells of the dorsal pancreatic bud,and separating said PDX1-positive, dorsally-biased, foregut endodermcells from other cell types present in the cell population.

120. The method of paragraph 119, wherein said enriched cell populationcomprises at least about 95% PDX1-positive, dorsally-biased, foregutendoderm cells.

121. The method of paragraph 119, wherein said enriched cell populationcomprises at least about 98% PDX1-positive, dorsally-biased, foregutendoderm cells.

122. The method of paragraph 119, wherein the differentiating stepfurther comprises, providing said pluripotent cell population with atleast one growth factor of the TGFβ superfamily in an amount sufficientto promote differentiation of said pluripotent cells to PDX1-negativedefinitive endoderm cells, and providing said PDX1-negative definitiveendoderm cells with a retinoid in an amount sufficient to promotedifferentiation of said PDX1-negative definitive endoderm cells toPDX1-positive, dorsally-biased, foregut endoderm cells.

123. The method of paragraph 122, wherein said retinoid is RA.

124. The method of paragraph 123, wherein the providing step furthercomprises providing B27.

125. The method of paragraph 119, wherein said fluorescent protein isgreen fluorescent protein (GFP).

126. The method of paragraph 119, wherein said PDX1-positive,dorsally-biased, foregut endoderm cells are separated from other celltypes present in the cell population by fluorescence activated cellsorting (FACS).

127. An enriched population of PDX1-positive, dorsally-biased, foregutendoderm cells produced by the method of paragraph 119.

128. A method of producing a cell population enriched in PDX1-positive,ventrally-biased, foregut endoderm cells, said method comprising thesteps of obtaining a population of pluripotent cells, wherein at leastone cell of said pluripotent cell population comprises at least one copyof a nucleic acid under the control of the a promoter of any one of themarker genes selected from Table 3, said nucleic acid comprising asequence encoding a fluorescent protein or a biologically activefragment thereof, differentiating said pluripotent cells so as toproduce PDX1-positive, ventrally-biased foregut endoderm cells, saidPDX1-positive, ventrally-biased, foregut endoderm cells beingmultipotent cells that can differentiate into cells of the ventralpancreatic bud and separating said PDX1-positive, ventrally-biased,foregut endoderm cells from non-ventrally-biased foregut endoderm cells.

129. The method of paragraph 128, wherein said enriched cell populationcomprises at least about 95% PDX1-positive, ventrally-biased, foregutendoderm cells.

130. The method of paragraph 128, wherein said enriched cell populationcomprises at least about 98% PDX1-positive, ventrally-biased, foregutendoderm cells.

131. The method of paragraph 128, wherein the differentiating stepfurther comprises, providing said pluripotent cell population with atleast one growth factor of the TGFβ superfamily in an amount sufficientto promote differentiation of said pluripotent cells to PDX1-negativedefinitive endoderm cells, and providing said PDX1-negative definitiveendoderm cells with an FGF-family growth factor in an amount sufficientto promote differentiation of said PDX1-negative definitive endodermcells to PDX1-positive, ventrally-biased, foregut endoderm cells.

132. The method of paragraph 128, wherein the providing step furthercomprises providing B27.

133. The method of paragraph 128, wherein said fluorescent protein isgreen fluorescent protein (GFP).

134. The method of paragraph 128, wherein said PDX1-positive,dorsally-biased, foregut endoderm cells are separated from other celltypes present in the cell population by fluorescence activated cellsorting (FACS).

135. An enriched population of PDX1-positive, dorsally-biased, foregutendoderm cells produced by the method of paragraph 128.

136. A method of identifying a differentiation factor capable ofpromoting the differentiation of human PDX1-positive, dorsally-biased,foregut endoderm cells in a cell population comprising human cells, saidmethod comprising the steps of obtaining a cell population comprisinghuman PDX1-positive, dorsally-biased, foregut endoderm cells, providinga candidate differentiation factor to said cell population, determiningexpression of a marker in said cell population at a first time point,determining expression of the same marker in said cell population at asecond time point, wherein said second time point is subsequent to saidfirst time point and wherein said second time point is subsequent toproviding said cell population with said candidate differentiationfactor and determining if expression of the marker in said cellpopulation at said second time point is increased or decreased ascompared to the expression of the marker in said cell population at saidfirst time point, wherein an increase or decrease in expression of saidmarker in said cell population indicates that said candidatedifferentiation factor is capable of promoting the differentiation ofsaid human PDX1-positive, dorsally-biased, foregut endoderm cells.

137. The method of paragraph 136, wherein said marker is selected fromTable 4.

138. The method of paragraph 136, wherein said human PDX1-positive,dorsally-biased, foregut endoderm cells comprise at least about 10% ofthe human cells in said cell population.

139. The method of paragraph 136, wherein human feeder cells are presentin said cell population and wherein at least about 10% of the humancells other than said feeder cells are PDX1-positive, dorsally-biased,foregut endoderm cells.

140. The method of paragraph 136, wherein said human PDX1-positive,dorsally-biased, foregut endoderm cells comprise at least about 90% ofthe human cells in said cell population.

141. The method of paragraph 136, wherein said human feeder cells arepresent in said cell population and wherein at least about 90% of thehuman cells other than said feeder cells are PDX1-positive,dorsally-biased, foregut endoderm cells.

142. The method of paragraph 136, wherein said human PDX1-positive,dorsally-biased, foregut endoderm cells can differentiate into cells ofthe dorsal pancreatic bud.

143. The method of paragraph 136, wherein said human definitive endodermcells differentiate into pancreatic precursor cells in response to saidcandidate differentiation factor.

144. The method of paragraph 136, wherein said first time point is priorto providing said candidate differentiation factor to said cellpopulation.

145. The method of paragraph 136, wherein said first time point is atapproximately the same time as providing said candidate differentiationfactor to said cell population.

146. The method of paragraph 136, wherein said first time point issubsequent to providing said candidate differentiation factor to saidcell population.

147. The method of paragraph 136, wherein expression of said marker isincreased.

148. The method of paragraph 136, wherein expression of said marker isdecreased.

149. The method of paragraph 136, wherein expression of said marker isdetermined by quantitative polymerase chain reaction (Q-PCR).

150. The method of paragraph 136, wherein expression of said marker isdetermined by immunocytochemistry.

151. The method of paragraph 136, wherein said differentiation factorcomprises a small molecule.

152. The method of paragraph 136, wherein said differentiation factorcomprises a polypeptide.

153. The method of paragraph 136, wherein said differentiation factorcomprises a growth factor.

154. A method of identifying a differentiation factor capable ofpromoting the differentiation of human PDX1-positive, ventrally-biased,foregut endoderm cells in a cell population comprising human cells, saidmethod comprising the steps of obtaining a cell population comprisinghuman PDX1-positive, ventrally-biased, foregut endoderm cells, providinga candidate differentiation factor to said cell population, determiningexpression of a marker in said cell population at a first time point,determining expression of the same marker in said cell population at asecond time point, wherein said second time point is subsequent to saidfirst time point and wherein said second time point is subsequent toproviding said cell population with said candidate differentiationfactor and determining if expression of the marker in said cellpopulation at said second time point is increased or decreased ascompared to the expression of the marker in said cell population at saidfirst time point, wherein an increase or decrease in expression of saidmarker in said cell population indicates that said candidatedifferentiation factor is capable of promoting the differentiation ofsaid human PDX1-positive, ventrally-biased, foregut endoderm cells.

155. The method of paragraph 154, wherein said marker is selected fromTable 3.

156. The method of paragraph 154, wherein said human PDX1-positive,ventrally-biased, foregut endoderm cells comprise at least about 10% ofthe human cells in said cell population.

157. The method of paragraph 154, wherein human feeder cells are presentin said cell population and wherein at least about 10% of the humancells other than said feeder cells are PDX1-positive, ventrally-biased,foregut endoderm cells.

158. The method of paragraph 154, wherein said human PDX1-positive,ventrally-biased, foregut endoderm cells comprise at least about 90% ofthe human cells in said cell population.

159. The method of paragraph 154, wherein said human feeder cells arepresent in said cell population and wherein at least about 90% of thehuman cells other than said feeder cells are PDX1-positive,ventrally-biased, foregut endoderm cells.

160. The method of paragraph 154, wherein said human PDX1-positive,ventrally-biased, foregut endoderm cells can differentiate into cells ofthe ventral pancreatic bud.

161. The method of paragraph 154, wherein said human definitive endodermcells differentiate into pancreatic precursor cells in response to saidcandidate differentiation factor.

162. The method of paragraph 154, wherein said first time point is priorto providing said candidate differentiation factor to said cellpopulation.

163. The method of paragraph 154, wherein said first time point is atapproximately the same time as providing said candidate differentiationfactor to said cell population.

164. The method of paragraph 154, wherein said first time point issubsequent to providing said candidate differentiation factor to saidcell population.

165. The method of paragraph 154, wherein expression of said marker isincreased.

166. The method of paragraph 154, wherein expression of said marker isdecreased.

167. The method of paragraph 154, wherein expression of said marker isdetermined by quantitative polymerase chain reaction (Q-PCR).

168. The method of paragraph 154, wherein expression of said marker isdetermined by immunocytochemistry.

169. The method of paragraph 154, wherein said differentiation factorcomprises a small molecule.

170. The method of paragraph 154, wherein said differentiation factorcomprises a polypeptide.

171. The method of paragraph 154, wherein said differentiation factorcomprises a growth factor.

It will be appreciated that the methods and compositions described aboverelate to cells cultured in vitro. However, the above-described in vitrodifferentiated cell compositions may be used for in vivo applications.

Additional embodiments of the present invention may also be found inU.S. Provisional Patent Application No. 60/532,004, entitled DEFINITIVEENDODERM, filed Dec. 23, 2003; U.S. Provisional Patent Application No.60/566,293, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 27, 2004; U.S.Provisional Patent Application No. 60/586,566, entitled CHEMOKINE CELLSURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9,2004; U.S. Provisional Patent Application No. 60/587,942, entitledCHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVEENDODERM, filed Jul. 14, 2004; U.S. patent application Ser. No.11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004 and U.S.patent application Ser. No. 11/115,868, entitled PDX1 EXPRESSINGENDODERM, filed Apr. 26, 2005; U.S. patent application Ser. No.11/165,305, entitled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATINGDEFINITIVE ENDODERM, filed Jun. 23, 2005, the disclosures of which areincorporated herein by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a proposed differentiation pathway for theproduction of beta-cells from hESCs. The first step in the pathwaycommits the ES cell to the definitive endoderm lineage and alsorepresents the first step prior to further differentiation events topancreatic endoderm, endocrine endoderm, or islet/beta-cells. The secondstep in the pathway shows the conversion of SOX17-positive/PDX1-negativedefinitive endoderm to PDX1-positive foregut endoderm. Some factorsuseful for mediating these transitions are italicized. Relevant markersfor defining the target cells are underlined.

FIG. 2 is a diagram of the human SOX17 cDNA which displays the positionsof conserved motifs and highlights the region used for the immunizationprocedure by GENOVAC.

FIG. 3 is a relational dendrogram illustrating that SOX17 is mostclosely related to SOX7 and somewhat less to SOX18. The SOX17 proteinsare more closely related among species homologs than to other members ofthe SOX group F subfamily within the same species.

FIG. 4 is a Western blot probed with the rat anti-SOX17 antibody. Thisblot demonstrates the specificity of this antibody for human SOX17protein over-expressed in fibroblasts (lane 1) and a lack ofimmunoreactivity with EGFP (lane 2) or the most closely related SOXfamily member, SOX7 (lane 3).

FIGS. 5A-B are micrographs showing a cluster of SOX17⁺ cells thatdisplay a significant number of AFP co-labeled cells (A). This is instriking contrast to other SOX17⁺ clusters (B) where little or no AFP⁺cells are observed.

FIGS. 6A-C are micrographs showing parietal endoderm and SOX17. Panel Ashows immunocytochemistry for human Thrombomodulin (TM) protein locatedon the cell surface of parietal endoderm cells in randomlydifferentiated cultures of hES cells. Panel B is the identical fieldshown in A double-labeled for TM and SOX17. Panel C is the phasecontrast image of the same field with DAPI labeled nuclei. Note thecomplete correlation of DAPI labeled nuclei and SOX17 labeling.

FIGS. 7A-B are bar charts showing SOX17 gene expression by quantitativePCR (Q-PCR) and anti-SOX17 positive cells by SOX17-specific antibody.Panel A shows that activin A increases SOX17 gene expression whileretinoic acid (RA) strongly suppresses SOX17 expression relative to theundifferentiated control media (SR20). Panel B shows the identicalpattern as well as a similar magnitude of these changes is reflected inSOX17⁺ cell number, indicating that Q-PCR measurement of SOX17 geneexpression is very reflective of changes at the single cell level.

FIG. 8A is a bar chart which shows that a culture of differentiatinghESCs in the presence of activin A maintains a low level of AFP geneexpression while cells allowed to randomly differentiate in 10% fetalbovine serum (FBS) exhibit a strong upregulation of AFP. The differencein expression levels is approximately 7-fold.

FIGS. 8B-C are images of two micrographs showing that the suppression ofAFP expression by activin A is also evident at the single cell level asindicated by the very rare and small clusters of AFP⁺ cells observed inactivin A treatment conditions (bottom) relative to 10% FBS alone (top).

FIGS. 9A-B are comparative images showing the quantitation of the AFP⁺cell number using flow cytometry. This figure demonstrates that themagnitude of change in AFP gene expression (FIG. 8A) in the presence(right panel) and absence (left panel) of activin A exactly correspondsto the number of AFP⁺ cells, further supporting the utility of Q-PCRanalyses to indicate changes occurring at the individual cell level.

FIGS. 10A-F are micrographs which show that exposure of hESCs to nodal,activin A and activin B (NAA) yields a striking increase in the numberof SOX17⁺ cells over the period of 5 days (A-C). By comparing to therelative abundance of SOX17⁺ cells to the total number of cells presentin each field, as indicated by DAPI stained nuclei (D-F), it can be seenthat approximately 30-50% of all cells are immunoreactive for SOX17after five days treatment with NAA.

FIG. 11 is a bar chart which demonstrates that activin A (0, 10, 30 or100 ng/ml) dose-dependently increases SOX17 gene expression indifferentiating hESCs. Increased expression is already robust after 3days of treatment on adherent cultures and continues through subsequent1, 3 and 5 days of suspension culture as well.

FIGS. 12A-C are bar charts which demonstrate the effect of activin A onthe expression of MIXL1 (panel A), GATA4 (panel B) and HNF3b (panel C).Activin A dose-dependent increases are also observed for three othermarkers of definitive endoderm; MIXL1, GATA4 and HNF3b. The magnitudesof increased expression in response to activin dose are strikinglysimilar to those observed for SOX17, strongly indicating that activin Ais specifying a population of cells that co-express all four genes(SOX17⁺, MIXL1⁺, GATA4⁺ and HNF3b⁺).

FIGS. 13A-C are bar charts which demonstrate the effect of activin A onthe expression of AFP (panel A), SOX7 (panel B) and SPARC (panel C).There is an activin A dose-dependent decrease in expression of thevisceral endoderm marker AFP. Markers of primitive endoderm (50×7) andparietal endoderm (SPARC) remain either unchanged or exhibit suppressionat some time points indicating that activin A does not act to specifythese extra-embryonic endoderm cell types. This further supports thefact that the increased expression of SOX17, MIXL1, GATA4, and HNF3b aredue to an increase in the number of definitive endoderm cells inresponse to activin A.

FIGS. 14A-B are bar charts showing the effect of activin A on ZIC1(panel A) and Brachyury expression (panel B) Consistent expression ofthe neural marker ZIC1 demonstrates that there is not a dose-dependenteffect of activin A on neural differentiation. There is a notablesuppression of mesoderm differentiation mediated by 100 ng/ml of activinA treatment as indicated by the decreased expression of brachyury. Thisis likely the result of the increased specification of definitiveendoderm from the mesendoderm precursors. Lower levels of activin Atreatment (10 and 30 ng/ml) maintain the expression of brachyury atlater time points of differentiation relative to untreated controlcultures.

FIGS. 15A-B are micrographs showing decreased parietal endodermdifferentiation in response to treatment with activins. Regions ofTM^(hi) parietal endoderm are found through the culture (A) whendifferentiated in serum alone, while differentiation to TM⁺ cells isscarce when activins are included (B) and overall intensity of TMimmunoreactivity is lower.

FIGS. 16A-D are micrographs which show marker expression in response totreatment with activin A and activin B. hESCs were treated for fourconsecutive days with activin A and activin B and triple labeled withSOX17, AFP and TM antibodies. Panel A—SOX17; Panel B—AFP; Panel C—TM;and Panel D—Phase/DAPI. Notice the numerous SOX17 positive cells (A)associated with the complete absence of AFP (B) and TM (C)immunoreactivity.

FIG. 17 is a micrograph showing the appearance of definitive endodermand visceral endoderm in vitro from hESCs. The regions of visceralendoderm are identified by AFP^(hi)/SOX17^(lo/−) while definitiveendoderm displays the complete opposite profile, SOX17^(hi)/AFP^(lo/−).This field was selectively chosen due to the proximity of these tworegions to each other. However, there are numerous times whenSOX17^(hi)/AFP^(lo/−) regions are observed in absolute isolation fromany regions of AFP^(hi) cells, suggesting the separate origination ofthe definitive endoderm cells from visceral endoderm cells.

FIG. 18 is a diagram depicting the TGFβ family of ligands and receptors.Factors activating AR Smads and BR Smads are useful in the production ofdefinitive endoderm from human embryonic stem cells (see, J CellPhysiol. 187:265-76).

FIG. 19 is a bar chart showing the induction of SOX17 expression overtime as a result of treatment with individual and combinations of TGFβfactors.

FIG. 20 is a bar chart showing the increase in SOX17⁺ cell number withtime as a result of treatment with combinations of TGFβ factors.

FIG. 21 is a bar chart showing induction of SOX17 expression over timeas a result of treatment with combinations of TGFβ factors.

FIG. 22 is a bar chart showing that activin A induces a dose-dependentincrease in SOX17⁺ cell number.

FIG. 23 is a bar chart showing that addition of Wnt3a to activin A andactivin B treated cultures increases SOX17 expression above the levelsinduced by activin A and activin B alone.

FIGS. 24A-C are bar charts showing differentiation to definitiveendoderm is enhanced in low FBS conditions. Treatment of hESCs withactivins A and B in media containing 2% FBS (2AA) yields a 2-3 timesgreater level of SOX17 expression as compared to the same treatment in10% FBS media (10AA) (panel A). Induction of the definitive endodermmarker MIXL1 (panel B) is also affected in the same way and thesuppression of AFP (visceral endoderm) (panel C) is greater in 2% FBSthan in 10% FBS conditions.

FIGS. 25A-D are micrographs which show SOX17⁺ cells are dividing inculture. SOX17 immunoreactive cells are present at the differentiatingedge of an hESC colony (C, D) and are labeled with proliferating cellnuclear antigen (PCNA) (panel B) yet are not co-labeled with OCT4 (panelC). In addition, clear mitotic figures can be seen by DAPI labeling ofnuclei in both SOX17⁺ cells (arrows) as well as OCT4⁺, undifferentiatedhESCs (arrowheads) (D).

FIG. 26 is a bar chart showing the relative expression level of CXCR4 indifferentiating hESCs under various media conditions.

FIGS. 27A-D are bar charts that show how a panel of definitive endodermmarkers share a very similar pattern of expression to CXCR4 across thesame differentiation treatments displayed in FIG. 26.

FIGS. 28A-E are bar charts showing how markers for mesoderm (BRACHYURY,MOX1), ectoderm (SOX1, ZIC1) and visceral endoderm (SOX7) exhibit aninverse relationship to CXCR4 expression across the same treatmentsdisplayed in FIG. 26.

FIGS. 29A-F are micrographs that show the relative difference in SOX17immunoreactive cells across three of the media conditions displayed inFIGS. 26-28.

FIGS. 30A-C are flow cytometry dot plots that demonstrate the increasein CXCR4⁺ cell number with increasing concentration of activin A addedto the differentiation media.

FIGS. 31A-D are bar charts that show the CXCR4⁺ cells isolated from thehigh dose activin A treatment (A100-CX+) are even further enriched fordefinitive endoderm markers than the parent population (A100).

FIG. 32 is a bar chart showing gene expression from CXCR4⁺ and CXCR4⁻cells isolated using fluorescence-activated cell sorting (FACS) as wellas gene expression in the parent populations. This demonstrates that theCXCR4⁺ cells contain essentially all the CXCR4 gene expression presentin each parent population and the CXCR4⁻ populations contain very littleor no CXCR4 gene expression.

FIGS. 33A-D are bar charts that demonstrate the depletion of mesoderm(BRACHYURY, MOX1), ectoderm (ZIC1) and visceral endoderm (SOX7) geneexpression in the CXCR4+ cells isolated from the high dose activin Atreatment which is already suppressed in expression of thesenon-definitive endoderm markers.

FIGS. 34A-M are bar charts showing the expression patterns of markergenes that can be used to identify definitive endoderm cells. Theexpression analysis of definitive endoderm markers, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1 is shown in panels G-L, respectively. Theexpression analysis of previously described lineage marking genes,SOX17, SOX7, SOX17/SOX7, TM, ZIC1, and MOX1 is shown in panels A-F,respectively. Panel M shows the expression analysis of CXCR4. Withrespect to each of panels A-M, the column labeled hESC indicates geneexpression from purified human embryonic stem cells; 2NF indicates cellstreated with 2% FBS, no activin addition; 0.1A100 indicates cellstreated with 0.1% FBS, 100 ng/ml activin A; 1A100 indicates cellstreated with 1% FBS, 100 ng/ml activin A; and 2A100 indicates cellstreated with 2% FBS, 100 ng/ml activin A.

FIG. 35 is a chart which shows the relative expression of the PDX1 genein a culture of hESCs after 4 days and 6 days with and without activinin the presence of retinoic acid (RA) and fibroblast growth factor(FGF-10) added on day 4.

FIGS. 36A-F are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 6 days with and withoutactivin in the presence of retinoic acid (RA) and fibroblast growthfactor (FGF-10) added on day 4. The panels show the relative levels ofexpression of the following marker genes: (A) SOX17; (B) SOX7; (C) AFP;(D) SOX1; (E) ZIC1; and (F) NFM.

FIGS. 37A-C are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 8 days with and withoutactivin in the presence or absence of combinations of retinoic acid(RA), fibroblast growth factor (FGF-10) and fibroblast growth factor(FGF-4) added on day 4. The panels show the relative levels ofexpression of the following marker genes: (A) PDX1; (B) SOX7; and (C)NFM.

FIGS. 38A-G are charts which show the relative expression of markergenes in a culture of definitive endoderm cells contacted with 50 ng/mlFGF-10 in combination with either 1 μM, 0.2 μM or 0.04 μM retinoic acid(RA) added on day 4. The panels show the relative levels of expressionof the following marker genes: (A) PDX1; (B) HOXA3; (C) HOXC6; (D)HOXA13; (E) CDX1; (F) SOX1; and (G) NFM.

FIGS. 39A-E are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 8 days with and withoutactivin in the presence of combinations of retinoic acid (RA),fibroblast growth factor (FGF-10) and one of the following: serumreplacement (SR), fetal bovine serum (FBS) or B27. The panels show therelative levels of expression of the following marker genes: (A) PDX1;(B) SOX7; (C) AFP; (D) ZIC1; and (E) NFM.

FIGS. 40A-B are charts which show the relative expression of markergenes for pancreas (PDX1, HNF6) and liver (HNF6) in a culture of hESCsafter 6 days (just prior to addition of RA) and at 9 days (three daysafter exposure to RA). Various conditions were included to compare theaddition of activin B at doses of 10 ng/ml (a10), 25 ng/ml (a25) or 50ng/ml (a50) in the presence of either 25 ng/ml (A25) or 50 ng/ml (A50)activin A. The condition without any activin A or activin B (NF) servesas the negative control for definitive endoderm and PDX1-positiveendoderm production. The panels show the relative levels of expressionof the following marker genes: (A) PDX1 and (B) HNF6.

FIGS. 41A-C are charts which show the relative expression of markergenes in a culture of hESCs with 100 ng/ml (A100), 50 ng/ml (A50) orwithout (NF) activin A at 5 days (just prior to retinoic acid addition)and at 2, 4, and 6 days after RA exposure (day 7, 9, and 11,respectively). The percentage label directly under each bar indicatesthe FBS dose during days 3-5 of differentiation. Starting at day 7,cells treated with RA (R) were grown in RPMI medium comprising 0.5% FBS.The RA concentration was 2 μM on day 7, 1 μM on day 9 and 0.2 μM on day11. The panels show the relative levels of expression of the followingmarker genes: (A) PDX1; (B) ZIC1; (C) SOX7.

FIGS. 42A-B are charts which show the relative expression of markergenes in a culture of hESCs treated first with activin A in low FBS toinduce definitive endoderm (day 5) and then with fresh (A25R) mediumcomprising 25 ng/ml activin A and RA or various conditioned media(MEFCM, CM#2, CM#3 and CM#4) and RA to induce PDX1-expressing endoderm.Marker expression was determined on days 5, 6, 7, 8 and 9. The panelsshow the relative levels of expression of the following marker genes:(A) PDX1; (B) CDX1.

FIG. 43 is a chart which shows the relative expression of PDX1 in aculture of hESCs treated first with activin A in low FBS to inducedefinitive endoderm and followed by fresh media comprising activin A andretinoic acid (A25R) or varying amounts of RA in conditioned mediadiluted into fresh media. Total volume of media is 5 ml in all cases.

FIG. 44 is a Western blot showing PDX1 immunoprecipitated fromRA-treated definitive endoderm cells 3 days (d8) and 4 days (d9) afterthe addition of RA and 50 ng/ml activin A.

FIG. 45 is a summary chart displaying the results of afluorescence-activated cell sort (FACs) of PDX1-positive foregutendoderm cells genetically tagged with a EGFP reporter under control ofthe PDX1 promoter.

FIG. 46 is a chart showing relative PDX1 expression levels normalized tohousekeeping genes for sorted populations of live cells (Live),EGFP-negative cells (Neg) and EGFP-positive cells (GFP+).

FIG. 47 is a chart showing relative PDX1 expression levels normalized tohousekeeping genes for sorted populations of live cells (Live),EGFP-negative cells (Neg), the half of the EGFP-positive cell populationthat has the lowest EGFP signal intensity (Lo) and the half of theEGFP-positive cell population that has the highest EGFP signal intensity(Hi).

FIGS. 48A-E are a charts showing the relative expression levelsnormalized to housekeeping genes of five pancreatic endoderm markers insorted populations of live cells (Live), EGFP-negative cells (Neg) andEGFP-positive cells (GFP+). Panels: A—NKX2.2; B—GLUT2; C—HNF3β; D—KRT19and E—HNF4α.

FIG. 49 are a charts showing the relative expression levels normalizedto housekeeping genes of two non-pancreatic endoderm markers in sortedpopulations of live cells (Live), EGFP-negative cells (Neg) andEGFP-positive cells (GFP+). Panels: A—ZIC1 and B—GFAP.

FIGS. 50A-D are charts showing the relative expression of marker genesin a culture of hESCs at the start of differentiation prior to additionof any factor (0 d), and either in the presence of 100 ng/ml activin A(A100) for 3 days (3 d) followed by 3 ng/ml BMP4 and 50 ng/ml FGF-10(B3F50) on days 4-11 (7 d, 9 d and 11 d) or in the presence of 100 ng/mlactivin A (A100) for 5 days (5 d) followed by 25 ng/ml activin A and 2μM RA (A25R) on day 6 and 7 (7 d). The panels show the relative levelsof expression of the following marker genes: (A) ALB; (B) PDX1; (C) HB9and (D) HHEX.

FIGS. 51A-D are charts showing the relative expression of marker genesin a culture of hESCs at the start of differentiation prior to additionof any factor (0 d), and after four days of differentiation under one ofthe four following conditions: (a) 100 ng/ml BMP4 and 5 μM SU5402(B/SU); (b) no factor (NF); (c) 15 ng/ml activin A (A15); or (d) 100ng/ml activin A. This differentiation is followed by incubation in thepresence of 3 ng/ml BMP4, 50 ng/ml FGF-10 and 0.5 μM KAAD-cyclopamine(B3F50K0.5) on days 5-12 (6 d, 8 d, 10 d and 12 d). The panels show therelative levels of expression of the following marker genes: (A) CER;(B) SOX17; (C) PDX1 and (D) ALB.

FIGS. 52A-B are charts showing the relative expression of marker genesin a culture of hESCs at the start of differentiation prior to additionof any factor (0 d); in the presence of 100 ng/ml activin A (A100) for 3days (3 d); in the presence of no factor (NF), 50 ng/ml FGF-10 and 0.5μM KAAD-cyclopamine (FK) or 3 ng/ml BMP4, 50 ng/ml FGF-10 and 0.5 μMKAAD-cyclopamine (BFK) on days 4 and 5 (5 d); and in the presence of nofactor in RPMI medium (2R), no factor in RPMI medium supplemented withB27 (ORB), no factor in CMRL medium supplemented with B27 (0CB), 50ng/ml FGF-10 and 0.5 μM KAAD-cyclopamine in CMRL medium supplementedwith B27 (FK) or 3 ng/ml BMP4, 50 ng/ml FGF-10 and 0.5 μMKAAD-cyclopamine in CMRL medium supplemented with B27 (BFK) on days 6-13(7 d, 9 d, 11 d and 13 d). The panels show the relative levels ofexpression of the following marker genes: (A) PDX1 and (B) ALB.

FIGS. 53A-E are charts showing the relative expression of marker genesin a culture of hESCs at the start of differentiation prior to additionof any factor (0 d), and either in the presence of 100 ng/ml activin A(A100) for 3 days (3 d) followed by 3 ng/ml BMP4 and 50 ng/ml FGF-10(B3F50) on days 4-11 (7 d, 9 d and 11 d) or in the presence of 100 ng/mlactivin A (A100) for 5 days (5 d) followed by 25 ng/ml activin A and 2μM RA (A25R) on day 6 and 7 (7 d). The panels show the relative levelsof expression of the following marker genes: (A) PDX1; (B) SERPINF2; (C)DUSP9; (D) CDH6 and (E) SOX9.

FIGS. 54A-D are charts showing the relative expression of marker genesin a culture of hESCs at the start of differentiation prior to additionof any factor (0 d), and either in the presence of 100 ng/ml activin A(A100) for 3 days (3 d) followed by 3 ng/ml BMP4 and 50 ng/ml FGF-10(B3F50) on days 4-11 (7 d, 9 d and 11 d) or in the presence of 100 ng/mlactivin A (A100) for 5 days (5 d) followed by 25 ng/ml activin A and 2μM RA (A25R) on day 6 and 7 (7 d). The panels show the relative levelsof expression of the following marker genes: (A) HOXA1; (B) PDE11A; (C)FAM49A and (D) WNT5A.

DETAILED DESCRIPTION

A crucial stage in early human development termed gastrulation occurs2-3 weeks after fertilization. Gastrulation is extremely significantbecause it is at this time that the three primary germ layers are firstspecified and organized (Lu et al., 2001; Schoenwolf and Smith, 2000).The ectoderm is responsible for the eventual formation of the outercoverings of the body and the entire nervous system whereas the heart,blood, bone, skeletal muscle and other connective tissues are derivedfrom the mesoderm. Definitive endoderm is defined as the germ layer thatis responsible for formation of the entire gut tube which includes theesophagus, stomach and small and large intestines, and the organs whichderive from the gut tube such as the lungs, liver, thymus, parathyroidand thyroid glands, gall bladder and pancreas (Grapin-Botton and Melton,2000; Kimelman and Griffin, 2000; Tremblay et al., 2000; Wells andMelton, 1999; Wells and Melton, 2000). A very important distinctionshould be made between the definitive endoderm and the completelyseparate lineage of cells termed primitive endoderm. The primitiveendoderm is primarily responsible for formation of extra-embryonictissues, mainly the parietal and visceral endoderm portions of theplacental yolk sac and the extracellular matrix material of Reichert'smembrane.

During gastrulation, the process of definitive endoderm formation beginswith a cellular migration event in which mesendoderm cells (cellscompetent to form mesoderm or endoderm) migrate through a structurecalled the primitive streak. Definitive endoderm is derived from cells,which migrate through the anterior portion of the streak and through thenode (a specialized structure at the anterior-most region of thestreak). As migration occurs, definitive endoderm populates first themost anterior gut tube and culminates with the formation of theposterior end of the gut tube.

The PDX1 Gene Expression During Development

PDX1 (also called STF-1, IDX-1, IPF-1, IUF-1 and GSF) is a transcriptionfactor that is necessary for development of the pancreas and rostralduodenum. PDX1 is first expressed in the pancreatic endoderm, whicharises from posterior foregut endoderm and will produce both theexocrine and endocrine cells, starting at E8.5 in the mouse. Later, PDX1becomes restricted to beta-cells and some delta-cells. This expressionpattern is maintained in the adult. PDX1 is also expressed in duodenalendoderm early in development, which is adjacent to the formingpancreas, then in the duodenal enterocytes and enteroendocrine cells,antral stomach and in the common bile, cystic and biliary ducts. Thisregion of expression also becomes limited, at the time that pancreaticexpression becomes restricted, to predominantly the rostral duodenum.

Targeted disruption of the PDX1 gene in mouse and man leads topancreatic agenesis (Jonsson, J., et al., Nature, 606-609, 1994;Offield, M F, et al. Devel. 983-995, 1996; Stoffers, D. A., et al.Nature Genetics, 106-110, 1997). PDX1 is also required during terminaldifferentiation of the insulin and somatostatin pancreatic endocrinecells and functional disruption of a single allele in humans isassociated with severe pancreatic dysfunction of MODY type 4 (maturityonset diabetes of the young) and late onset type II diabetes (StoffersD. A., et al. Nature Genetics 138-139, 1997).

During embryogenesis of the pancreas a budding of the prospectivepancreatic tissue occurs on both the dorsal and ventral side of theprimitive gut endoderm. These protrusions occur in a regionally definedmanner at the most posterior end of the foregut endoderm. In mice thisoccurs at approximately 8.5-9.5 days post conception (dpc) and in humansat 30 dpc. By 35 dpc in the human, the ventral and dorsal buds havegrown, developed a branched ductal system and fused to form thedefinitive organ. PDX1 protein is required for the early pancreatic budsto expand and differentiate into the principal cells comprising thepancreas which include duct, acinar and endocrine cells.

Due to their locations on opposite sides of the gut tube and theirrespective associations with notochord and cardiac mesoderm thedevelopmental programs for the dorsal and ventral pancreatic structuresare distinctly different. With regard to the unique developmentalprograms controlling specification of the dorsal and ventral pancreaticanlaga we have developed two separate methodologies for producingdorsally-biased and ventrally-biased PDX1-expressing foregut endodermfrom definitive endoderm (DE) cultures generated from human embryonicstem cells (hESCs). These PDX1-expressing (PDX1-positive) foregutendoderm cells are competent to develop into pancreatic and duodenalepithelium as well as endocrine cells of the anterior gastric mucosa.

Aspects of the present invention relate to the discovery that definitiveendoderm cells can be differentiated into at least two distinguishabletypes of PDX1-expressing (PDX1-positive) foregut endoderm cells. We havealso discovered that prior to the expression of PDX1, definitiveendoderm cells can be differentiated into a PDX1-negative foregutendoderm cell. Providing these PDX1-negative cells with a retinoidcompound, such as retinoic acid, induces the expression of PDX1. Inanother aspect of the present invention, definitive endoderm cells aredifferentiated to form dorsal PDX1-positive foregut endoderm cells. Asused herein, with respect to PDX1-positive foregut endoderm, “dorsal” or“dorsally-biased” means that the PDX1-positive foregut endoderm cellsare those that can give rise to tissues derived from the dorsal side ofthe posterior portion of the foregut, such as the dorsal pancreatic bud.Once a PDX1-positive foregut endoderm cell becomes “dorsally-biased” itdoes not typically develop into tissues derived from the ventral side ofthe posterior portion of the foregut. In another aspect, definitiveendoderm cells are differentiated to form ventral PDX1-positive foregutendoderm cells. As used herein, with respect to PDX1-positive foregutendoderm, “ventral” or “ventrally-biased” means that the PDX1-positiveforegut endoderm cells are those that can give rise to tissues derivedfrom the ventral side of the posterior portion of the foregut, such asthe liver and the ventral pancreatic bud. Once a PDX1-positive foregutendoderm cell becomes “ventrally-biased” it does not typically developinto tissues derived from the dorsal side of the posterior portion ofthe foregut.

In view of the foregoing discovery, embodiments of the present inventionrelate to compositions of PDX1-negative foregut endoerm cells,dorsally-biased PDX1-positive foregut endoderm cells, ventrally-biasedPDX1-positive foregut endoderm cells and/or compositions comprisingmixtures of dorsally-biased and ventrally-biased PDX1-positive foregutendoderm cells as well as methods for the production of suchcompositions. Other embodiments of the present invention relate toscreening of PDX1-negative foregut endoderm cells for factors thatpromote the differentiation of such cells. Still other embodimentsrelate to screening dorsal, ventral or mixed populations ofPDX1-positive foregut endoderm cells for factors that promote thedifferentiation of such cells. By “mixed populations” is meant a cellpopulation comprising significant amounts of both dorsal PDX1-positiveforegut endoderm cells and ventral PDX1-positive foregut endoderm cells.

As used herein, FGF-family growth factor includes, but is not limitedto, FGF-family growth factor selected from the group consisting of FGF1,FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12,FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22and/or FGF23.

As used herein, hedgehog inhibitor includes, but is not limited to,KKAD-cyclopamine, KKAD-cyclopamine analogs, jervine, jervine analogs,hedgehog pathway blocking antibodies and any other inhibitors ofhedgehog pathway function known to those of ordinary skill in the art.

PDX1-Negative Foregut Endoderm Cells and Processes Related Thereto

Embodiments of the present invention relate to novel, defined processesfor the production of PDX1-negative endoderm cells, wherein thePDX1-negative endoderm cells are multipotent cells that candifferentiate into cells, tissues or organs derived from theforegut/midgut region of the gut tube (PDX1-negative foregut/midgutendoderm). As used herein, “multipotent” or “multipotent cell” refers toa cell type that can give rise to a limited number of other particularcell types but which cannot give rise to all three primary embryoniccells lineages (endoderm, ectoderm and mesoderm). As used herein,“foregut/midgut” refers to cells of the anterior portion of the gut tubeas well as cells of the middle portion of the gut tube, including cellsof the foregut/midgut junction.

Some preferred embodiments of the present invention relate to processesfor the production of PDX1-negative foregut endoderm cells. In someembodiments, these PDX1-negative foregut endoderm cells are multipotentcells that can differentiate into cells, tissues or organs derived fromthe anterior portion of the gut tube (PDX1-negative foregut endoderm).

Dorsal, Ventral and Mixed Populations of PDX1-Positive Foregut EndodermCells and Processes Related Thereto

Embodiments of the present invention relate to novel, defined processesfor the production of PDX1-negative endoderm cells, wherein thePDX1-positive endoderm cells are multipotent cells that candifferentiate into cells, tissues or organs derived from theforegut/midgut region of the gut tube (PDX1-positive foregut/midgutendoderm). Other embodiments of the present invention relate to novel,defined processes for the production of PDX1-positive endoderm cells,wherein the PDX1-positive endoderm cells are multipotent cells that candifferentiate into cells, tissues or organs derived from theforegut/midgut region of the gut tube (PDX1-positive foregut/midgutendoderm). As used herein, “multipotent” or “multipotent cell” refers toa cell type that can give rise to a limited number of other particularcell types but which cannot give rise to all three primary embryoniccells lineages (endoderm, ectoderm and mesoderm). As used herein,“foregut/midgut” refers to cells of the anterior portion of the gut tubeas well as cells of the middle portion of the gut tube, including cellsof the foregut/midgut junction. In some embodiments, the PDX1 positiveendoderm cells are dorsal foregut endoderm cells. In other embodiments,the PDX1 positive endoderm cells are ventral foregut endoderm cells.

Some preferred embodiments of the present invention relate to processesfor the production of PDX1-positive foregut endoderm cells. In someembodiments, these PDX1-positive foregut endoderm cells are multipotentcells that can differentiate into cells, tissues or organs derived fromthe anterior portion of the gut tube (PDX1-positive foregut endoderm).In some embodiments, the PDX1 positive endoderm cells are dorsal foregutendoderm cells. In other embodiments, the PDX1 positive endoderm cellsare ventral foregut endoderm cells.

Additional preferred embodiments relate to processes for the productionof PDX1-positive endoderm cells of the posterior portion of the foregut.In some embodiments, these PDX1-positive endoderm cells are multipotentcells that can differentiate into cells, tissues or organs derived fromthe posterior portion of the foregut region of the gut tube. In someembodiments, the PDX1 positive endoderm cells are dorsal endoderm cellsthat can differentiate into cells, tissues or organs derived from theposterior portion of the foregut, such as cells of the dorsal pancreaticbud. In other embodiments, the PDX1 positive endoderm cells are ventralforegut endoderm cells that can differentiate into cells, tissues ororgans derived from the posterior portion of the foregut, such as cellsof the ventral pancreatic bud.

The dorsal and/or ventral PDX1-positive foregut endoderm cells, such asthose produced according to the methods described herein, can be used toproduce fully differentiated insulin-producing β-cells. In someembodiments of the present invention, positive dorsal and/or ventralPDX1-foregut endoderm cells are produced by differentiating definitiveendoderm cells that do not substantially express PDX1 (PDX1-negativedefinitive endoderm cells; also referred to herein as definitiveendoderm) so as to form positive dorsal and/or ventral PDX1-positiveforegut endoderm cells. PDX1-negative definitive endoderm cells can beprepared by differentiating pluripotent cells, such as embryonic stemcells, as described herein or by any other known methods. A convenientand highly efficient method for producing PDX1-negative definitiveendoderm from pluripotent cells is described in U.S. patent Ser. No.11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, thedisclosure of which is incorporated herein by reference in its entirety.

Processes of producing PDX1-positive foregut endoderm cells, includingdorsal and/or ventral PDX1-positive foregut endoderm cells, provide abasis for efficient production of pancreatic tissues such as acinarcells, ductal cells and islet cells from pluripotent cells. In certainpreferred embodiments, human dorsal and/or ventral PDX1-positive foregutendoderm cells are derived from human PDX1-negative definitive endodermcells, which in turn, are derived from hESCs. These human dorsal and/orventral PDX1-positive foregut endoderm cells can then be used to producefunctional insulin-producing β-cells. To obtain useful quantities ofinsulin-producing β-cells, high efficiency of differentiation isdesirable for each of the differentiation steps that occur prior toreaching the pancreatic islet/β-cell fate. Because differentiation ofPDX1-negative definitive endoderm cells to dorsal and/or ventralPDX1-positive foregut endoderm cells represents an early step towardsthe production of functional pancreatic islet/β-cells (as shown in FIG.1), high efficiency of differentiation at this step is particularlydesirable.

In view of the desirability of efficient differentiation ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells, some aspects of the present invention relate to in vitromethodology that results in approximately 2-25% conversion ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells. Some aspects of the present invention relate to in vitromethodology that results in approximately 26% to at least approximately75% conversion of PDX1-negative definitive endoderm cells to dorsalPDX1-positive foregut endoderm cells. Other aspects of the presentinvention relate to in vitro methodology that results in approximately26% to at least approximately 75% conversion of PDX1-negative definitiveendoderm cells to ventral PDX1-positive foregut endoderm cells.Typically, the above-described methods encompass the application ofculture and growth factor conditions in a defined and temporallyspecified fashion. Further enrichment of the cell population forPDX1-positive foregut endoderm cells, including dorsal and/or ventralPDX1-positive foregut endoderm cells, can be achieved by isolationand/or purification of the PDX1-positive foregut endoderm cells fromother cells in the population by using a reagent that specifically bindsto the PDX1-positive foregut endoderm cells. As an alternative,PDX1-positive foregut endoderm cells, including dorsal and/or ventralPDX1-positive foregut endoderm cells, can be labeled with a reportergene, such as green fluorescent protein (GFP), so as to enable thedetection of PDX1 expression. Such fluorescently labeled cells can thenbe purified by fluorescent activated cell sorting (FACS). Furtheraspects of the present invention relate to cell cultures and enrichedcell populations comprising PDX1-positive foregut endoderm cells as wellas methods for identifying factors useful in the differentiation to andfrom PDX1-positive foregut endoderm. Additional aspects of the presentinvention relate to cell cultures and enriched cell populationscomprising dorsal PDX1-positive foregut endoderm cells as well asmethods for identifying factors useful in the differentiation to andfrom dorsal PDX1-positive foregut endoderm. Still other aspects of thepresent invention relate to cell cultures and enriched cell populationscomprising ventral PDX1-positive foregut endoderm cells as well asmethods for identifying factors useful in the differentiation to andfrom ventral PDX1-positive foregut endoderm.

In order to determine the amount of PDX1-positive foregut endoderm cellsin a cell culture or cell population, a method of distinguishing thiscell type from the other cells in the culture or in the population isdesirable. Accordingly, certain embodiments of the present inventionrelate to cell markers whose presence, absence and/or relativeexpression levels are indicative of PDX1-positive foregut endodermcells, including dorsal and/or ventral PDX1-positive foregut endodermcells, as well as methods for detecting and determining the expressionof such markers. As used herein, “expression” refers to the productionof a material or substance as well as the level or amount of productionof a material or substance. Thus, determining the expression of aspecific marker refers to detecting either the relative or absoluteamount of the marker that is expressed or simply detecting the presenceor absence of the marker. As used herein, “marker” refers to anymolecule that can be observed or detected. For example, a marker caninclude, but is not limited to, a nucleic acid, such as a transcript ofa specific gene, a polypeptide product of a gene, a non-gene productpolypeptide, a glycoprotein, a carbohydrate, a glycolipd, a lipid, alipoprotein or a small molecule (for example, molecules having amolecular weight of less than 10,000 amu).

In some embodiments of the present invention, the presence, absenceand/or level of expression of a marker is determined by quantitative PCR(Q-PCR). For example, the amount of transcript produced by certaingenetic markers, such as PDX1, SOX17, SOX7, SOX1, ZIC1, NFM,alpha-fetoprotein (AFP), homeobox A13 (HOXA13), homeobox C6 (HOXC6),and/or other markers described herein is determined by Q-PCR. In otherembodiments, immunohistochemistry is used to detect the proteinsexpressed by the above-mentioned genes. In still other embodiments,Q-PCR and immunohistochemical techniques are both used to identify anddetermine the amount or relative proportions of such markers. In someembodiments, markers that are common to both dorsal and ventralPDX1-positive foregut endoderm cells, such as PDX1 and/or one or moremarkers selected from Table 3, are detected by Q-PCR and/orimmunohistochemistry. In other embodiments, markers that arepreferentially, specifically or uniquely expressed in dorsalPDX1-positive foregut endoderm cells, such as one or more markersselected from Table 4, are detected by Q-PCR and/orimmunohistochemistry.

By using the differentiation and detection methods described herein, itis possible to identify PDX1-positive foregut endoderm cells, includingdorsal and/or ventral PDX1-positive foregut endoderm cells, as well asdetermine the proportion of dorsal and/or ventral PDX1-positive foregutendoderm cells in a cell culture or cell population. For example, insome embodiments of the present invention, the dorsal and/or ventralPDX1-positive foregut endoderm cells or cell populations that areproduced express the PDX1 gene at a level of at least about 2 orders ofmagnitude greater than PDX1-negative cells or cell populations. In otherembodiments, the dorsal and/or ventral PDX1-positive foregut endodermcells and cell populations that are produced express the PDX1 gene at alevel of more than 2 orders of magnitude greater than PDX1-negativecells or cell populations. In still other embodiments, the dorsal and/orventral PDX1-positive foregut endoderm cells or cell populations thatare produced express one or more of the markers selected from the groupconsisting of PDX1, SOX17, HOXA13 and HOXC6 at a level of about 2 ormore than 2 orders of magnitude greater than PDX1-negative definitiveendoderm cells or cell populations. In yet other embodiments, the dorsaland/or ventral PDX1-positive foregut endoderm cells or cell populationsthat are produced express one or more of the markers selected from Table3 at a level of about 2 or more than 2 orders of magnitude greater thanPDX1-negative definitive endoderm cells or cell populations. In furtherembodiments, the dorsal PDX1-positive foregut endoderm cells or cellpopulations that are produced express one or more of the markersselected from Table 4 at a level of about 2 or more than 2 orders ofmagnitude greater than PDX1-negative definitive endoderm cells or cellpopulations.

The compositions and methods described herein have several usefulfeatures. For example, the cell cultures and cell populations comprisingPDX1-positive endoderm, including dorsal and/or ventral PDX1-positiveforegut endoderm, as well as the methods for producing such cellcultures and cell populations, are useful for modeling the early stagesof human development. Furthermore, the compositions and methodsdescribed herein can also serve for therapeutic intervention in diseasestates, such as diabetes mellitus. For example, since PDX1-positiveforegut endoderm serves as the source for only a limited number oftissues, it can be used in the development of pure tissue or cell types.

Production of PDX1-Negative Definitive Endoderm (Definitive Endoderm)from Pluripotent Cells

Cell cultures and/or cell populations comprising PDX1-positive foregutendoderm cells are produced from pluripotent cells by first producingPDX1-negative definitive endoderm (also referred to as “definitiveendoderm”). Processes for differentiating pluripotent cells to producecell cultures and enriched cell populations comprising definitiveendoderm is described briefly below and in detail in U.S. patent Ser.No. 11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, thedisclosure of which is incorporated herein by reference in its entirety.In some of these processes, the pluripotent cells used as startingmaterial are stem cells. In certain processes, definitive endoderm cellcultures and enriched cell populations comprising definitive endodermcells are produced from embryonic stem cells. As used herein,“embryonic” refers to a range of developmental stages of an organismbeginning with a single zygote and ending with a multicellular structurethat no longer comprises pluripotent or totipotent cells other thandeveloped gametic cells. In addition to embryos derived by gametefusion, the term “embryonic” refers to embryos derived by somatic cellnuclear transfer. A preferred method for deriving definitive endodermcells utilizes human embryonic stem cells as the starting material fordefinitive endoderm production. Such pluripotent cells can be cells thatoriginate from the morula, embryonic inner cell mass or those obtainedfrom embryonic gonadal ridges. Human embryonic stem cells can bemaintained in culture in a pluripotent state without substantialdifferentiation using methods that are known in the art. Such methodsare described, for example, in U.S. Pat. Nos. 5,453,357, 5,670,372,5,690,926 5,843,780, 6,200,806 and 6,251,671 the disclosures of whichare incorporated herein by reference in their entireties.

In some processes for producing definitive endoderm cells, hESCs aremaintained on a feeder layer. In such processes, any feeder layer whichallows hESCs to be maintained in a pluripotent state can be used. Onecommonly used feeder layer for the cultivation of human embryonic stemcells is a layer of mouse fibroblasts. More recently, human fibroblastfeeder layers have been developed for use in the cultivation of hESCs(see US Patent Application No. 2002/0072117, the disclosure of which isincorporated herein by reference in its entirety). Alternative processesfor producing definitive endoderm permit the maintenance of pluripotenthESC without the use of a feeder layer. Methods of maintainingpluripotent hESCs under feeder-free conditions have been described in USPatent Application No. 2003/0175956, the disclosure of which isincorporated herein by reference in its entirety.

The human embryonic stem cells used herein can be maintained in cultureeither with or without serum. In some embryonic stem cell maintenanceprocedures, serum replacement is used. In others, serum free culturetechniques, such as those described in US Patent Application No.2003/0190748, the disclosure of which is incorporated herein byreference in its entirety, are used.

Stem cells are maintained in culture in a pluripotent state by routinepassage until it is desired that they be differentiated into definitiveendoderm. In some processes, differentiation to definitive endoderm isachieved by providing to the stem cell culture a growth factor of theTGFβ superfamily in an amount sufficient to promote differentiation todefinitive endoderm. Growth factors of the TGFβ superfamily which areuseful for the production of definitive endoderm are selected from theNodal/Activin or BMP subgroups. In some preferred differentiationprocesses, the growth factor is selected from the group consisting ofNodal, activin A, activin B and BMP4. Additionally, the growth factorWnt3a and other Wnt family members are useful for the production ofdefinitive endoderm cells. In certain differentiation processes,combinations of any of the above-mentioned growth factors can be used.

With respect to some of the processes for the differentiation ofpluripotent stem cells to definitive endoderm cells, the above-mentionedgrowth factors are provided to the cells so that the growth factors arepresent in the cultures at concentrations sufficient to promotedifferentiation of at least a portion of the stem cells to definitiveendoderm cells. In some processes, the above-mentioned growth factorsare present in the cell culture at a concentration of at least about 5ng/ml, at least about 10 ng/ml, at least about 25 ng/ml, at least about50 ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at leastabout 200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, atleast about 500 ng/ml, at least about 1000 ng/ml, at least about 2000ng/ml, at least about 3000 ng/ml, at least about 4000 ng/ml, at leastabout 5000 ng/ml or more than about 5000 ng/ml.

In certain processes for the differentiation of pluripotent stem cellsto definitive endoderm cells, the above-mentioned growth factors areremoved from the cell culture subsequent to their addition. For example,the growth factors can be removed within about one day, about two days,about three days, about four days, about five days, about six days,about seven days, about eight days, about nine days or about ten daysafter their addition. In a preferred processes, the growth factors areremoved about four days after their addition.

Cultures of definitive endoderm cells can be grown in medium containingreduced serum or no serum. Under certain culture conditions, serumconcentrations can range from about 0.05% v/v to about 20% v/v. Forexample, in some differentiation processes, the serum concentration ofthe medium can be less than about 0.05% (v/v), less than about 0.1%(v/v), less than about 0.2% (v/v), less than about 0.3% (v/v), less thanabout 0.4% (v/v), less than about 0.5% (v/v), less than about 0.6%(v/v), less than about 0.7% (v/v), less than about 0.8% (v/v), less thanabout 0.9% (v/v), less than about 1% (v/v), less than about 2% (v/v),less than about 3% (v/v), less than about 4% (v/v), less than about 5%(v/v), less than about 6% (v/v), less than about 7% (v/v), less thanabout 8% (v/v), less than about 9% (v/v), less than about 10% (v/v),less than about 15% (v/v) or less than about 20% (v/v). In someprocesses, definitive endoderm cells are grown without serum or withserum replacement. In still other processes, definitive endoderm cellsare grown in the presence of B27. In such processes, the concentrationof B27 supplement can range from about 0.1% v/v to about 20% v/v.

Monitoring the Differentiation of Pluripotent Cells to PDX1-NegativeDefinitive Endoderm (Definitive Endoderm)

The progression of the hESC culture to definitive endoderm can bemonitored by determining the expression of markers characteristic ofdefinitive endoderm. In some processes, the expression of certainmarkers is determined by detecting the presence or absence of themarker. Alternatively, the expression of certain markers can bedetermined by measuring the level at which the marker is present in thecells of the cell culture or cell population. In such processes, themeasurement of marker expression can be qualitative or quantitative. Onemethod of quantitating the expression of markers that are produced bymarker genes is through the use of quantitative PCR (Q-PCR). Methods ofperforming Q-PCR are well known in the art. Other methods which areknown in the art can also be used to quantitate marker gene expression.For example, the expression of a marker gene product can be detected byusing antibodies specific for the marker gene product of interest. Incertain processes, the expression of marker genes characteristic ofdefinitive endoderm as well as the lack of significant expression ofmarker genes characteristic of hESCs and other cell types is determined.

As described further in the Examples below, a reliable marker ofdefinitive endoderm is the SOX17 gene. As such, the definitive endodermcells produced by the processes described herein express the SOX17marker gene, thereby producing the SOX17 gene product. Other markers ofdefinitive endoderm are MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1. Since definitive endoderm cells express theSOX17 marker gene at a level higher than that of the SOX7 marker gene,which is characteristic of primitive and visceral endoderm (see Table1), in some processes, the expression of both SOX17 and SOX7 ismonitored. In other processes, expression of the both the SOX17 markergene and the OCT4 marker gene, which is characteristic of hESCs, ismonitored. Additionally, because definitive endoderm cells express theSOX17 marker gene at a level higher than that of the AFP, SPARC orThrombomodulin (TM) marker genes, the expression of these genes can alsobe monitored.

Another marker of definitive endoderm is the CXCR4 gene. The CXCR4 geneencodes a cell surface chemokine receptor whose ligand is thechemoattractant SDF-1. The principal roles of the CXCR4 receptor-bearingcells in the adult are believed to be the migration of hematopoeticcells to the bone marrow, lymphocyte trafficking and the differentiationof various B cell and macrophage blood cell lineages [Kim, C., andBroxmeyer, H. J. Leukocyte Biol. 65, 6-15 (1999)]. The CXCR4 receptoralso functions as a coreceptor for the entry of HIV-1 into T-cells[Feng, Y., et al. Science, 272, 872-877 (1996)]. In an extensive seriesof studies carried out by [McGrath, K. E. et al. Dev. Biology 213,442-456 (1999)], the expression of the chemokine receptor CXCR4 and itsunique ligand, SDF-1 [Kim, C., and Broxmyer, H., J. Leukocyte Biol. 65,6-15 (1999)], were delineated during early development and adult life inthe mouse. The CXCR4/SDF1 interaction in development became apparentwhen it was demonstrated that if either gene was disrupted in transgenicmice [Nagasawa et al. Nature, 382, 635-638 (1996)], Ma, Q., et alImmunity, 10, 463-471 (1999)] it resulted in late embryonic lethality.McGrath et al. demonstrated that CXCR4 is the most abundant chemokinereceptor messenger RNA detected during early gastrulating embryos (E7.5)using a combination of RNase protection and in situ hybridizationmethodologies. In the gastrulating embryo, CXCR4/SDF-1 signaling appearsto be mainly involved in inducing migration of primitive-streakgermlayer cells and is expressed on definitive endoderm, mesoderm andextraembryonic mesoderm present at this time. In E7.2-7.8 mouse embryos,CXCR4 and alpha-fetoprotein are mutually exclusive indicating a lack ofexpression in visceral endoderm [McGrath, K. E. et al. Dev. Biology 213,442-456 (1999)].

Since definitive endoderm cells produced by differentiating pluripotentcells express the CXCR4 marker gene, expression of CXCR4 can bemonitored in order to track the production of definitive endoderm cells.Additionally, definitive endoderm cells produced by the methodsdescribed herein express other markers of definitive endoderm including,but not limited to, SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1. Since definitive endoderm cells express theCXCR4 marker gene at a level higher than that of the SOX7 marker gene,the expression of both CXCR4 and SOX7 can be monitored. In otherprocesses, expression of the both the CXCR4 marker gene and the OCT4marker gene, is monitored. Additionally, because definitive endodermcells express the CXCR4 marker gene at a level higher than that of theAFP, SPARC or Thrombomodulin (TM) marker genes, the expression of thesegenes can also be monitored.

It will be appreciated that expression of CXCR4 in endodermal cells doesnot preclude the expression of SOX17. As such, definitive endoderm cellsproduced by the processes described herein will substantially expressSOX17 and CXCR4 but will not substantially express AFP, TM, SPARC orPDX1.

Enrichment, Isolation and/or Purification of Definitive Endoderm

Definitive endoderm cells produced by any of the above-describedprocesses can be enriched, isolated and/or purified by using an affinitytag that is specific for such cells. Examples of affinity tags specificfor definitive endoderm cells are antibodies, ligands or other bindingagents that are specific to a marker molecule, such as a polypeptide,that is present on the cell surface of definitive endoderm cells butwhich is not substantially present on other cell types that would befound in a cell culture produced by the methods described herein. Insome processes, an antibody which binds to CXCR4 is used as an affinitytag for the enrichment, isolation or purification of definitive endodermcells. In other processes, the chemokine SDF-1 or other molecules basedon SDF-1 can also be used as affinity tags. Such molecules include, butnot limited to, SDF-1 fragments, SDF-1 fusions or SDF-1 mimetics.

Methods for making antibodies and using them for cell isolation areknown in the art and such methods can be implemented for use with theantibodies and definitive endoderm cells described herein. In oneprocess, an antibody which binds to CXCR4 is attached to a magnetic beadand then allowed to bind to definitive endoderm cells in a cell culturewhich has been enzymatically treated to reduce intercellular andsubstrate adhesion. The cell/antibody/bead complexes are then exposed toa movable magnetic field which is used to separate bead-bound definitiveendoderm cells from unbound cells. Once the definitive endoderm cellsare physically separated from other cells in culture, the antibodybinding is disrupted and the cells are replated in appropriate tissueculture medium.

Additional methods for obtaining enriched, isolated or purifieddefinitive endoderm cell cultures or populations can also be used. Forexample, in some embodiments, the CXCR4 antibody is incubated with adefinitive endoderm-containing cell culture that has been treated toreduce intercellular and substrate adhesion. The cells are then washed,centrifuged and resuspended. The cell suspension is then incubated witha secondary antibody, such as an FITC-conjugated antibody that iscapable of binding to the primary antibody. The cells are then washed,centrifuged and resuspended in buffer. The cell suspension is thenanalyzed and sorted using a fluorescence activated cell sorter (FACS).CXCR4-positive cells are collected separately from CXCR4-negative cells,thereby resulting in the isolation of such cell types. If desired, theisolated cell compositions can be further purified by using an alternateaffinity-based method or by additional rounds of sorting using the sameor different markers that are specific for definitive endoderm.

In still other processes, definitive endoderm cells are enriched,isolated and/or purified using a ligand or other molecule that binds toCXCR4. In some processes, the molecule is SDF-1 or a fragment, fusion ormimetic thereof.

In preferred processes, definitive endoderm cells are enriched, isolatedand/or purified from other non-definitive endoderm cells after the stemcell cultures are induced to differentiate towards the definitiveendoderm lineage. It will be appreciated that the above-describedenrichment, isolation and purification procedures can be used with suchcultures at any stage of differentiation.

In addition to the procedures just described, definitive endoderm cellsmay also be isolated by other techniques for cell isolation.Additionally, definitive endoderm cells may also be enriched or isolatedby methods of serial subculture in growth conditions which promote theselective survival or selective expansion of the definitive endodermcells.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of definitive endoderm cells and or tissues can be producedin vitro from pluripotent cell cultures or cell populations, such asstem cell cultures or populations, which have undergone at least somedifferentiation. In some methods, the cells undergo randomdifferentiation. In a preferred method, however, the cells are directedto differentiate primarily into definitive endoderm. Some preferredenrichment, isolation and/or purification methods relate to the in vitroproduction of definitive endoderm from human embryonic stem cells. Usingthe methods described herein, cell populations or cell cultures can beenriched in definitive endoderm content by at least about 2- to about1000-fold as compared to untreated cell populations or cell cultures.

Compositions Comprising PDX1-Negative Definitive Endoderm (DefinitiveEndoderm)

Cell compositions produced by the above-described methods include cellcultures comprising definitive endoderm and cell populations enriched indefinitive endoderm. For example, cell cultures which comprisedefinitive endoderm cells, wherein at least about 50-80% of the cells inculture are definitive endoderm cells, can be produced. Because theefficiency of the differentiation process can be adjusted by modifyingcertain parameters, which include but are not limited to, cell growthconditions, growth factor concentrations and the timing of culturesteps, the differentiation procedures described herein can result inabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orgreater than about 95% conversion of pluripotent cells to definitiveendoderm. In processes in which isolation of definitive endoderm cellsis employed, for example, by using an affinity reagent that binds to theCXCR4 receptor, a substantially pure definitive endoderm cell populationcan be recovered.

Production of PDX1-Negative Foregut Endoderm

Definitive endoderm cells can be specified toward pancreaticdifferentiation by further differentiation of these cells to producePDX1-negative foregut endoderm cells. In some of the differentiationprocesses described herein, cell cultures as well as enriched orpurified cell populations comprising definitive endoderm cells can beused for further differentiation to cell cultures and/or enriched cellpopulations comprising PDX1-negative foregut endoderm cells.

Typically, definitive endoderm cells are differentiated to PDX1-negativeforegut endoderm cells by reducing or eliminating TGFβ superfamilygrowth factor signaling in a cell culture or cell population ofSOX17-positive definitive endoderm cells. In some embodiments, reducingor eliminating TGFβ superfamily growth factor signaling is mediated bydiluting or removing an exogenously added TGFβ superfamily growthfactor, such as activin A, from the cell culture or cell population ofdefinitive endoderm. In other embodiments, TGFβ superfamily growthfactor signaling is reduced or eliminated by providing the definitiveendoderm cells with a compound that blocks TGFβ superfamily growthfactor signaling, such as follistatin and/or noggin. In someembodiments, TGFβ superfamily growth factor signaling can be reduced oreliminated for about one day, about two days, about three days, aboutfour days, about five days, about six days, about seven days, abouteight days, about nine days, about ten days or greater than about tendays subsequent to the differentiation of the human pluripotent cells todefinitive endoderm cells.

In some embodiments, differentiation of definitive endoderm cells toforegut endoderm cells is enhanced by providing the definitive endodermcell culture or cell population with an FGF-family growth factor and/ora hedgehog pathway inhibitor. In such embodiments the FGF-family growthfactor and/or hedgehog pathway inhibitor is provided at about one day,about two days, about three days, about four days, about five days,about six days, about seven days, about eight days, about nine days,about ten days or greater than about ten days subsequent to reducing oreliminating TGFβ superfamily growth factor signaling in the definitiveendoderm cell culture. In a preferred embodiment, the FGF-family growthfactor and/or hedgehog pathway inhibitor is provided at about the sametime as reducing or eliminating TGFβ superfamily growth factor signalingin the definitive endoderm cell culture.

In a preferred embodiment, the FGF-family growth factor provided to thedefinitive endoderm cell culture or cell population is FGF10 and/orFGF7. However, it will be appreciated that other FGF-family growthfactors or FGF-family growth factor analogs or mimetics may be providedinstead of or in addition to FGF10 and/or FGF7. For example, anFGF-family growth factor selected from the group consisting of FGF1,FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12,FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22and/or FGF23 may be provided. In such embodiments, the FGF-family growthfactor and/or the FGF-family growth factor analog or mimetic is providedto the cells of a cell culture such that it is present at aconcentration of at least about 10 ng/ml, at least about 25 ng/ml, atleast about 50 ng/ml, at least about 75 ng/ml, at least about 100 ng/ml,at least about 200 ng/ml, at least about 300 ng/ml, at least about 400ng/ml, at least about 500 ng/ml, or at least about 1000 ng/ml.

In other preferred embodiments, the hedgehog inhibitor isKAAD-cyclopamine. However, it will be appreciated that other hedgehoginhibitors can be used. Such inhibitors include, but are not limited to,KKAD-cyclopamine analogs, jervine, jervine analogs, hedgehog pathwayblocking antibodies and any other inhibitors of hedgehog pathwayfunction known to those of ordinary skill in the art. When used alone orin conjunction with FGF-family growth factor, the hedgehog inhibitor canbe provided at a concentration of at least about 0.01 μM, at least about0.02 μM, at least about 0.04 μM, at least about 0.08 μM, at least about0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4μM, at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM,at least about 0.8 μM, at least about 0.9 μM, at least about 1 μM, atleast about 1.1 μM, at least about 1.2 μM, at least about 1.3 μM, atleast about 1.4 μM, at least about 1.5 μM, at least about 1.6 μM, atleast about 1.7 μM, at least about 1.8 μM, at least about 1.9 μM, atleast about 2 μM, at least about 2.1 μM, at least about 2.2 μM, at leastabout 2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at leastabout 2.6 μM, at least about 2.7 μM, at least about 2.8 μM, at leastabout 2.9 μM, at least about 3 μM, at least about 3.5 μM, at least about4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM,at least about 20 μM, at least about 30 μM, at least about 40 μM or atleast about 50 μM.

In a preferred process for the production of a population ofPDX1-negative foregut endoderm cells from definitive endoderm cells,TGFβ superfamily growth factor signaling is reduced or eliminated forabout two day subsequent to the differentiation of a substantial portionof human pluripotent cells to definitive endoderm (for example, after athree day, four or five day differentiation protocol as described in theexamples below). At about the same time, the cell culture or cellpopulation of definitive endoderm cells is provided with 50 ng/ml ofFGF-10 and 0.2 μM KAAD-cyclopamine.

Cultures of PDX1-negative foregut endoderm cells can be differentiatedand further grown in a medium containing reduced or no serum. Serumconcentrations can range from about 0.05% (v/v) to about 20% (v/v). Insome processes, PDX1-negative foregut endoderm cells are grown withserum replacement. For example, in certain processes, the serumconcentration of the medium can be less than about 0.05% (v/v), lessthan about 0.1% (v/v), less than about 0.2% (v/v), less than about 0.3%(v/v), less than about 0.4% (v/v), less than about 0.5% (v/v), less thanabout 0.6% (v/v), less than about 0.7% (v/v), less than about 0.8%(v/v), less than about 0.9% (v/v), less than about 1% (v/v), less thanabout 2% (v/v), less than about 3% (v/v), less than about 4% (v/v), lessthan about 5% (v/v), less than about 6% (v/v), less than about 7% (v/v),less than about 8% (v/v), less than about 9% (v/v), less than about 10%(v/v), less than about 15% (v/v) or less than about 20% (v/v). Incertain processes described herein, the differentiation medium does notinclude serum, serum replacement or any supplement comprising insulin orinsulin-like growth factors.

In certain processes, PDX1-negative foregut endoderm cells are grown inthe presence of B27. In such differentiation processes, B27 can beprovided to the culture medium in concentrations ranging from about 0.1%(v/v) to about 20% (v/v) or in concentrations greater than about 20%(v/v). In certain processes, the concentration of B27 in the medium isabout 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v),about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v),about 0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about4% (v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8%(v/v), about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20%(v/v). Alternatively, the concentration of the added B27 supplement canbe measured in terms of multiples of the strength of a commerciallyavailable B27 stock solution. For example, B27 is available fromInvitrogen (Carlsbad, Calif.) as a 50× stock solution. Addition of asufficient amount of this stock solution to a sufficient volume ofgrowth medium produces a medium supplemented with the desired amount ofB27. For example, the addition of 10 ml of 50×B27 stock solution to 90ml of growth medium would produce a growth medium supplemented with5×B27. The concentration of B27 supplement in the medium can be about0.1×, about 0.2×, about 0.3×, about 0.4×, about 0.5×, about 0.6×, about0.7×, about 0.8×, about 0.9×, about 1×, about 1.1×, about 1.2×, about1.3×, about 1.4×, about 1.5×, about 1.6×, about 1.7×, about 1.8×, about1.9×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×,about 5×, about 6×, about 7×, about 8×, about 9×, about 10×, about 11×,about 12×, about 13×, about 14×, about 15×, about 16×, about 17×, about18×, about 19×, about 20× and greater than about 20×.

In some embodiments, the PDX1-negative foregut endoderm cells can befurther differentiated to PDX1-positive foregut endoderm cells bycontacting the cells with a medium comprising, or otherwise providing tothe cells, a retinoid, such as retinoic acid (RA). In some embodiments,the retinoid is provided to the cells of a cell culture such that it ispresent at a concentration of at least about 1 nM, at least about 0.01μM, at least about 0.02 μM, at least about 0.04 μM, at least about 0.08μM, at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM,at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, atleast about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM, atleast about 1 μM, at least about 1.1 μM, at least about 1.2 μM, at leastabout 1.3 μM, at least about 1.4 μM, at least about 1.5 μM, at leastabout 1.6 μM, at least about 1.7 μM, at least about 1.8 μM, at leastabout 1.9 μM, at least about 2 μM, at least about 2.1 μM, at least about2.2 μM, at least about 2.3 μM, at least about 2.4 μM, at least about 2.5μM, at least about 2.6 μM, at least about 2.7 μM, at least about 2.8 μM,at least about 2.9 μM, at least about 3 μM, at least about 3.5 μM, atleast about 4 μM, at least about 4.5 μM, at least about 5 μM, at leastabout 10 μM, at least about 20 μM, at least about 30 μM, at least about40 μM or at least about 50 μM. In such embodiments, the retinoid isprovided to the cells at about one day, about two days, about threedays, about four days, about five days, about six days, about sevendays, about eight days, about nine days, about ten days or greater thanabout ten days subsequent to reducing or eliminating TGFβ superfamilygrowth factor signaling in the definitive endoderm cell culture. In apreferred embodiment, from about 0.05 μM RA to about 2 μM RA is providedto the PDX-1 negative foregut endoderm cell culture about 2 to 3 dayssubsequent to reducing or eliminating TGFβ superfamily growth factorsignaling.

In some of the differentiation processes described herein, theabove-mentioned differentiation factors are removed from the cellculture subsequent to their addition. For example, the above-mentioneddifferentiation factors can be removed within about one day, about twodays, about three days, about four days, about five days, about sixdays, about seven days, about eight days, about nine days or about tendays after their addition.

Monitoring the Differentiation of PDX1-Negative Definitive Endoderm toPDX1-Negative Foregut Endoderm

Expression of HNF1b and/or FOXA1 and the lack of expression of PDX1 canbe detected and/or quantitated using the above-described methods, suchas Q-PCR and/or immunocytochemistry, to monitor the differentiation ofPDX1-negative definitive endoderm to PDX1-negative foregut endoderm. Inaddition to the above-described markers, in some embodiments of thepresent invention, the expression of SOX17 is also determined

In some embodiments, PDX1-negative foregut endoderm cell culturesproduced by the methods described herein are substantially free of cellsexpressing the SOX7, AFP, SOX1, ZIC1 or NFM marker genes. In certainembodiments, the PDX1-negative foregut endoderm cell cultures producedby the processes described herein are substantially free of visceralendoderm, parietal endoderm and/or neural cells.

Compositions Comprising PDX1-Negative Foregut Endoderm

Some embodiments of the present invention relate to cell compositions,such as cell cultures or cell populations, comprising PDX1-negativeforegut endoderm cells, wherein the PDX1-negative foregut endoderm cellsare multipotent cells that can differentiate into cells, tissues ororgans derived from the anterior portion of the gut tube. In accordancewith certain embodiments, the PDX1-negative foregut endoderm cells aremammalian cells, and in a preferred embodiment, such cells are humancells.

Other embodiments of the present invention relate to compositions, suchas cell cultures or cell populations, comprising cells of one or morecell types selected from the group consisting of hESCs, PDX1-negativedefinitive endoderm cells, PDX1-negative foregut endoderm cells andmesoderm cells. In some embodiments, hESCs comprise less than about 5%,less than about 4%, less than about 3%, less than about 2% or less thanabout 1% of the total cells in the culture. In other embodiments,PDX1-negative definitive endoderm cells comprise less than about 90%,less than about 85%, less than about 80%, less than about 75%, less thanabout 70%, less than about 65%, less than about 60%, less than about55%, less than about 50%, less than about 45%, less than about 40%, lessthan about 35%, less than about 30%, less than about 25%, less thanabout 20%, less than about 15%, less than about 12%, less than about10%, less than about 8%, less than about 6%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2% or less than about1% of the total cells in the culture. In yet other embodiments, mesodermcells comprise less than about 90%, less than about 85%, less than about80%, less than about 75%, less than about 70%, less than about 65%, lessthan about 60%, less than about 55%, less than about 50%, less thanabout 45%, less than about 40%, less than about 35%, less than about30%, less than about 25%, less than about 20%, less than about 15%, lessthan about 12%, less than about 10%, less than about 8%, less than about6%, less than about 5%, less than about 4%, less than about 3%, lessthan about 2% or less than about 1% of the total cells in the culture.

Additional embodiments of the present invention relate to compositions,such as cell cultures or cell populations, produced by the processesdescribed herein comprise PDX1-negative foregut endoderm as the majoritycell type. In some embodiments, the processes described herein producecell cultures and/or cell populations comprising at least about 99%, atleast about 98%, at least about 97%, at least about 96%, at least about95%, at least about 94%, at least about 93%, at least about 92%, atleast about 91%, at least about 90%, at least about 89%, at least about88%, at least about 87%, at least about 86%, at least about 85%, atleast about 84%, at least about 83%, at least about 82%, at least about81%, at least about 80%, at least about 79%, at least about 78%, atleast about 77%, at least about 76%, at least about 75%, at least about74%, at least about 73%, at least about 72%, at least about 71%, atleast about 70%, at least about 69%, at least about 68%, at least about67%, at least about 66%, at least about 65%, at least about 64%, atleast about 63%, at least about 62%, at least about 61%, at least about60%, at least about 59%, at least about 58%, at least about 57%, atleast about 56%, at least about 55%, at least about 54%, at least about53%, at least about 52%, at least about 51% or at least about 50%PDX1-negative foregut endoderm cells. In preferred embodiments the cellsof the cell cultures or cell populations comprise human cells. In otherembodiments, the processes described herein produce cell cultures orcell populations comprising at least about 50%, at least about 45%, atleast about 40%, at least about 35%, at least about 30%, at least about25%, at least about 24%, at least about 23%, at least about 22%, atleast about 21%, at least about 20%, at least about 19%, at least about18%, at least about 17%, at least about 16%, at least about 15%, atleast about 14%, at least about 13%, at least about 12%, at least about11%, at least about 10%, at least about 9%, at least about 8%, at leastabout 7%, at least about 6%, at least about 5%, at least about 4%, atleast about 3%, at least about 2% or at least about 1% PDX1-negativeforegut endoderm cells. In preferred embodiments, the cells of the cellcultures or cell populations comprise human cells. In some embodiments,the percentage of PDX1-negative foregut endoderm cells in the cellcultures or populations is calculated without regard to the feeder cellsremaining in the culture.

Still other embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising PDX1-negativeforegut endoderm cells and PDX1-negative definitive endoderm cells. Forexample, cell cultures or cell populations comprising at least about 5PDX1-negative foregut endoderm cells for about every 95 PDX1-negativedefinitive endoderm cells can be produced. In other embodiments, cellcultures or cell populations comprising at least about 95 PDX1-negativeforegut endoderm cells for about every 5 PDX1-negative definitiveendoderm cells can be produced. Additionally, cell cultures or cellpopulations comprising other ratios of PDX1-negative foregut endodermcells to PDX1-negative definitive endoderm cells are contemplated. Forexample, compositions comprising at least about 1 PDX1-negative foregutendoderm cell for about every 1,000,000 PDX1-negative definitiveendoderm cells, at least about 1 PDX1-negative foregut endoderm cell forabout every 100,000 PDX1-negative definitive endoderm cells, at leastabout 1 PDX1-negative foregut endoderm cell for about every 10,000PDX1-negative definitive endoderm cells, at least about 1 PDX1-negativeforegut endoderm cell for about every 1000 PDX1-negative definitiveendoderm cells, at least about 1 PDX1-negative foregut endoderm cell forabout every 500 PDX1-negative definitive endoderm cells, at least about1 PDX1-negative foregut endoderm cell for about every 100 PDX1-negativedefinitive endoderm cells, at least about 1 PDX1-negative foregutendoderm cell for about every 10 PDX1-negative definitive endodermcells, at least about 1 PDX1-negative foregut endoderm cell for aboutevery 5 PDX1-negative definitive endoderm cells, at least about 1PDX1-negative foregut endoderm cell for about every 4 PDX1-negativedefinitive endoderm cells, at least about 1 PDX1-negative foregutendoderm cell for about every 2 PDX1-negative definitive endoderm cells,at least about 1 PDX1-negative foregut endoderm cell for about every 1PDX1-negative definitive endoderm cell, at least about 2 PDX1-negativeforegut endoderm cells for about every 1 PDX1-negative definitiveendoderm cell, at least about 4 PDX1-negative foregut endoderm cells forabout every 1 PDX1-negative definitive endoderm cell, at least about 5PDX1-negative foregut endoderm cells for about every 1 PDX1-negativedefinitive endoderm cell, at least about 10 PDX1-negative foregutendoderm cells for about every 1 PDX1-negative definitive endoderm cell,at least about 20 PDX1-negative foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 50 PDX1-negativeforegut endoderm cells for about every 1 PDX1-negative definitiveendoderm cell, at least about 100 PDX1-negative foregut endoderm cellsfor about every 1 PDX1-negative definitive endoderm cell, at least about1000 PDX1-negative foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 10,000PDX1-negative foregut endoderm cells for about every 1 PDX1-negativedefinitive endoderm cell, at least about 100,000 PDX1-negative foregutendoderm cells for about every 1 PDX1-negative definitive endoderm celland at least about 1,000,000 PDX1-negative foregut endoderm cells forabout every 1 PDX1-negative definitive endoderm cell are contemplated.

In some embodiments of the present invention, the PDX1-negativedefinitive endoderm cells from which PDX1-negative foregut endodermcells are produced are derived from human pluripotent cells, such ashuman pluripotent stem cells. In certain embodiments, the humanpluripotent cells are derived from a morula, the inner cell mass of anembryo or the gonadal ridges of an embryo. In certain other embodiments,the human pluripotent cells are derived from the gonadal or germ tissuesof a multicellular structure that has developed past the embryonicstage.

Further embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising human cells,including human PDX1-negative foregut endoderm cells, wherein theexpression of the SOX17, HNF1b and/or FOXA1 marker is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 2% of the human cells. In other embodiments, the expression of theSOX17, HNF1b and/or FOXA1 marker is greater than the expression of theAFP, SOX7, SOX1, ZIC1 and/or NFM marker in at least about 5% of thehuman cells, in at least about 10% of the human cells, in at least about15% of the human cells, in at least about 20% of the human cells, in atleast about 25% of the human cells, in at least about 30% of the humancells, in at least about 35% of the human cells, in at least about 40%of the human cells, in at least about 45% of the human cells, in atleast about 50% of the human cells, in at least about 55% of the humancells, in at least about 60% of the human cells, in at least about 65%of the human cells, in at least about 70% of the human cells, in atleast about 75% of the human cells, in at least about 80% of the humancells, in at least about 85% of the human cells, in at least about 90%of the human cells, in at least about 95% of the human cells or in atleast about 98% of the human cells. In some embodiments, the percentageof human cells in the cell cultures or populations, wherein theexpression of SOX17, HNF1b and/or FOXA1 is greater than the expressionof the AFP, SOX7, SOX1, ZIC1 and/or NFM marker, is calculated withoutregard to feeder cells.

It will be appreciated that some embodiments of the present inventionrelate to compositions, such as cell cultures or cell populations,comprising human PDX1-negative foregut endoderm cells, wherein theexpression of one or more markers selected from the group consisting ofSOX17, HNF1b and/or FOXA1 is greater than the expression of the PDX1marker in from at least about 2% to greater than at least about 98% ofthe human cells. In some embodiments, the expression of one or moremarkers selected from the group consisting of SOX17, HNF1b and/or FOXA1is greater than the expression of the PDX1 marker in at least about 5%of the human cells, in at least about 10% of the human cells, in atleast about 15% of the human cells, in at least about 20% of the humancells, in at least about 25% of the human cells, in at least about 30%of the human cells, in at least about 35% of the human cells, in atleast about 40% of the human cells, in at least about 45% of the humancells, in at least about 50% of the human cells, in at least about 55%of the human cells, in at least about 60% of the human cells, in atleast about 65% of the human cells, in at least about 70% of the humancells, in at least about 75% of the human cells, in at least about 80%of the human cells, in at least about 85% of the human cells, in atleast about 90% of the human cells, in at least about 95% of the humancells or in at least about 98% of the human cells. In some embodiments,the percentage of human cells in the cell cultures or populations,wherein the expression of one or more markers selected from the groupconsisting of SOX17, HNF1b and/or FOXA1 is greater than the expressionof the PDX1 marker, is calculated without regard to feeder cells.

Using the processes described herein, compositions comprisingPDX1-negative foregut endoderm cells substantially free of other celltypes can be produced. With respect to cells in cell cultures or in cellpopulations, the term “substantially free of” means that the specifiedcell type of which the cell culture or cell population is free, ispresent in an amount of less than about 5% of the total number of cellspresent in the cell culture or cell population. In some embodiments ofthe present invention, the PDX1-negative foregut endoderm cellpopulations or cell cultures produced by the methods described hereinare substantially free of cells that significantly express the AFP,SOX7, SOX1, ZIC1 and/or NFM marker genes.

In one embodiment of the present invention, a description of aPDX1-negative foregut endoderm cell based on the expression of markergenes is, SOX17 high, HNF1b high, FOXA1 high, PDX1 low, AFP low, SOX7low, SOX1 low, ZIC1 low and NFM low.

Production of PDX1-Positive Foregut Endoderm Directly from PDX1-NegativeDefinitive Endoderm

The PDX1-positive foregut endoderm cell cultures and populationscomprising PDX1-positive foregut endoderm cells that are describedherein are produced from PDX1-negative definitive endoderm, which isgenerated from pluripotent cells as described above. A preferred methodutilizes human embryonic stem cells as the starting material. In oneembodiment, hESCs are first converted to PDX1-negative definitiveendoderm cells, which are then converted to PDX1-positive foregutendoderm cells. It will be appreciated, however, that the startingmaterials for the production of PDX1-positive foregut endoderm is notlimited to definitive endoderm cells produced using pluripotent celldifferentiation methods. Rather, any PDX1-negative definitive endodermcells can be used in the methods described herein regardless of theirorigin.

In some embodiments of the present invention, cell cultures or cellpopulations comprising PDX1-negative definitive endoderm cells can beused for further differentiation to cell cultures and/or enriched cellpopulations comprising PDX1-positive foregut endoderm cells. Forexample, a cell culture or cell population comprising humanPDX1-negative, SOX17-positive definitive endoderm cells can be used. Insome embodiments, the cell culture or cell population may also comprisedifferentiation factors, such as activins, nodals and/or BMPs, remainingfrom the previous differentiation step (that is, the step ofdifferentiating pluripotent cells to definitive endoderm cells). Inother embodiments, factors utilized in the previous differentiation stepare removed from the cell culture or cell population prior to theaddition of factors used for the differentiation of the PDX1-negative,SOX17-positive definitive endoderm cells to PDX1-positive foregutendoderm cells. In other embodiments, cell populations enriched forPDX1-negative, SOX17-positive definitive endoderm cells are used as asource for the production of PDX1-positive foregut endoderm cells.

PDX1-negative definitive endoderm cells in culture are differentiated toPDX1-positive endoderm cells by providing to a cell culture comprisingPDX1-negative, SOX17-positive definitive endoderm cells adifferentiation factor that promotes differentiation of the cells toPDX1-positive foregut endoderm cells (foregut differentiation factor).In some embodiments of the present invention, the foregutdifferentiation factor is retinoid, such as retinoic acid (RA). In someembodiments, the retinoid is used in conjunction with a fibroblastgrowth factor, such as FGF-4 or FGF-10. In other embodiments, theretinoid is used in conjunction with a member of the TGFβ superfamily ofgrowth factors and/or a conditioned medium.

By “conditioned medium” is meant, a medium that is altered as comparedto a base medium. For example, the conditioning of a medium may causemolecules, such as nutrients and/or growth factors, to be added to ordepleted from the original levels found in the base medium. In someembodiments, a medium is conditioned by allowing cells of certain typesto be grown or maintained in the medium under certain conditions for acertain period of time. For example, a medium can be conditioned byallowing hESCs to be expanded, differentiated or maintained in a mediumof defined composition at a defined temperature for a defined number ofhours. As will be appreciated by those of skill in the art, numerouscombinations of cells, media types, durations and environmentalconditions can be used to produce nearly an infinite array ofconditioned media. In some embodiments of the present invention, amedium is conditioned by allowing differentiated pluripotent cells to begrown or maintained in a medium comprising about 1% to about 20% serumconcentration. In other embodiments, a medium is conditioned by allowingdifferentiated pluripotent cells to be grown or maintained in a mediumcomprising about 1 ng/ml to about 1000 ng/ml activin A. In still otherembodiments, a medium is conditioned allowing differentiated pluripotentcells to be grown or maintained in a medium comprising about 1 ng/ml toabout 1000 ng/ml BMP4. In a preferred embodiment, a conditioned mediumis prepared by allowing differentiated hESCs to be grown or maintainedfor 24 hours in a medium, such as RPMI, comprising about 25 ng/mlactivin A and about 2 μM RA.

In some embodiments of the present invention, the cells used tocondition the medium, which is used to enhance the differentiation ofPDX1-negative definitive endoderm to PDX1-positive foregut endoderm, arecells that are differentiated from pluripotent cells, such as hESCs,over about a 5 day time period in a medium such as RPMI comprising about0% to about 20% serum and/or one or more growth/differentiation factorsof the TGFβ superfamily. Differentiation factors, such as activin A andBMP4 are supplied at concentrations ranging from about 1 ng/ml to about1000 ng/ml. In certain embodiments of the present invention, the cellsused to condition the medium are differentiated from hESCs over about a5 day period in low serum RPMI. According to some embodiments, low serumRPMI refers to a low serum containing medium, wherein the serumconcentration is gradually increased over a defined time period. Forexample, in one embodiment, low serum RPMI comprises a concentration ofabout 0.2% fetal bovine serum (FBS) on the first day of cell growth,about 0.5% FBS on the second day of cell growth and about 2% FBS on thethird through fifth day of cell growth. In another embodiment, low serumRPMI comprises a concentration of about 0% on day one, about 0.2% on daytwo and about 2% on days 3-6. In certain preferred embodiments, lowserum RPMI is supplemented with one or more differentiation factors,such as activin A and BMP4. In addition to its use in preparing cellsused to condition media, low serum RPMI can be used as a medium for thedifferentiation of PDX1-positive foregut endoderm cells fromPDX1-negative definitive endoderm cells.

It will be appreciated by those of ordinary skill in the art thatconditioned media can be prepared from media other than RPMI providedthat such media do not interfere with the growth or maintenance ofPDX1-positive foregut endoderm cells. It will also be appreciated thatthe cells used to condition the medium can be of various types. Inembodiments where freshly differentiated cells are used to condition amedium, such cells can be differentiated in a medium other than RPMIprovided that the medium does not inhibit the growth or maintenance ofsuch cells. Furthermore, a skilled artisan will appreciate that neitherthe duration of conditioning nor the duration of preparation of cellsused for conditioning is required to be 24 hours or 5 days,respectively, as other time periods will be sufficient to achieve theeffects reported herein.

In general, the use of a retinoid in combination with a fibroblastgrowth factor, a member of the TGFβ superfamily of growth factors, aconditioned medium or a combination of any of these foregutdifferentiation factors causes greater differentiation of PDX1-negativedefinitive endoderm to PDX1-positive foregut endoderm than the use of aretinoid alone. In a preferred embodiment, RA and FGF-10 are bothprovided to the PDX1-negative definitive endoderm cell culture. Inanother preferred embodiment, PDX1-negative definitive endoderm cellsare differentiated in a culture comprising a conditioned medium, activinA, activin B and RA.

With respect to some of the embodiments of differentiation processesdescribed herein, the above-mentioned foregut differentiation factorsare provided to the cells so that these factors are present in the cellculture or cell population at concentrations sufficient to promotedifferentiation of at least a portion of the PDX1-negative definitiveendoderm cell culture or cell population to PDX1-positive foregutendoderm cells. When used in connection with cell cultures and/or cellpopulations, the term “portion” means any non-zero amount of the cellculture or cell population, which ranges from a single cell to theentirety of the cell culture or cells population.

In some embodiments of the present invention, a retinoid is provided tothe cells of a cell culture such that it is present at a concentrationof at least about 0.01 μM, at least about 0.02 μM, at least about 0.04μM, at least about 0.08 μM, at least about 0.1 μM, at least about 0.2μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM,at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, atleast about 0.9 μM, at least about 1 μM, at least about 1.1 μM, at leastabout 1.2 μM, at least about 1.3 μM, at least about 1.4 μM, at leastabout 1.5 μM, at least about 1.6 μM, at least about 1.7 μM, at leastabout 1.8 μM, at least about 1.9 μM, at least about 2 μM, at least about2.1 μM, at least about 2.2 μM, at least about 2.3 μM, at least about 2.4μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM,at least about 2.8 μM, at least about 2.9 μM, at least about 3 μM, atleast about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at leastabout 5 μM, at least about 10 μM, at least about 20 μM, at least about30 μM, at least about 40 μM or at least about 50 μM. As used herein,“retinoid” refers to retinol, retinal or retinoic acid as well asderivatives of any of these compounds. In a preferred embodiment, theretinoid is retinoic acid.

In other embodiments of the present invention, one or moredifferentiation factors of the fibroblast growth factor family arepresent in the cell culture. For example, in some embodiments, FGF-4 canbe present in the cell culture at a concentration of at least about 10ng/ml, at least about 25 ng/ml, at least about 50 ng/ml, at least about75 ng/ml, at least about 100 ng/ml, at least about 200 ng/ml, at leastabout 300 ng/ml, at least about 400 ng/ml, at least about 500 ng/ml, orat least about 1000 ng/ml. In further embodiments of the presentinvention, FGF-10 is present in the cell culture at a concentration ofat least about 10 ng/ml, at least about 25 ng/ml, at least about 50ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at least about200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at leastabout 500 ng/ml, or at least about 1000 ng/ml. In some embodiments,either FGF-4 or FGF-10, but not both, is provided to the cell culturealong with RA. In a preferred embodiment, RA is present in the cellculture at 1 μM and FGF-10 is present at a concentration of 50 ng/ml.

In some embodiments of the present invention, growth factors of the TGFβsuperfamily and/or a conditioned medium are present in the cell culture.These differentiation factors can be used in combination with RA and/orother mid-foregut differentiation factors including, but not limited to,FGF-4 and FGF-10. For example, in some embodiments, activin A and/oractivin B can be present in the cell culture at a concentration of atleast about 5 ng/ml, at least about 10 ng/ml, at least about 25 ng/ml,at least about 50 ng/ml, at least about 75 ng/ml, at least about 100ng/ml, at least about 200 ng/ml, at least about 300 ng/ml, at leastabout 400 ng/ml, at least about 500 ng/ml, or at least about 1000 ng/ml.In further embodiments of the present invention, a conditioned medium ispresent in the cell culture at a concentration of at least about 1%, atleast about 5%, at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or at leastabout 100% of the total medium. In some embodiments, activin A, activinB and a conditioned medium are provided to the cell culture along withRA. In a preferred embodiment, PDX1-negative definitive endoderm cellsare differentiated to PDX1-positive foregut endoderm cells in culturescomprising about 1 μM RA, about 25 ng/ml activin A and low serum RPMImedium that has been conditioned for about 24 hours by differentiatedhESCs, wherein the differentiated hESCs have been differentiated forabout 5 days in low serum RPMI comprising about 100 ng/ml activin A. Inanother preferred embodiment, activin B and/or FGF-10 are also presentin the culture at 25 ng/ml and 50 ng/ml, respectively.

In certain embodiments of the present invention, the above-mentionedforegut differentiation factors are removed from the cell culturesubsequent to their addition. For example, the foregut differentiationfactors can be removed within about one day, about two days, about threedays, about four days, about five days, about six days, about sevendays, about eight days, about nine days or about ten days after theiraddition.

Cultures of PDX1-positive foregut endoderm cells can be grown in amedium containing reduced serum. Serum concentrations can range fromabout 0.05% (v/v) to about 20% (v/v). In some embodiments, PDX1-positiveforegut endoderm cells are grown with serum replacement. For example, incertain embodiments, the serum concentration of the medium can be lessthan about 0.05% (v/v), less than about 0.1% (v/v), less than about 0.2%(v/v), less than about 0.3% (v/v), less than about 0.4% (v/v), less thanabout 0.5% (v/v), less than about 0.6% (v/v), less than about 0.7%(v/v), less than about 0.8% (v/v), less than about 0.9% (v/v), less thanabout 1% (v/v), less than about 2% (v/v), less than about 3% (v/v), lessthan about 4% (v/v), less than about 5% (v/v), less than about 6% (v/v),less than about 7% (v/v), less than about 8% (v/v), less than about 9%(v/v), less than about 10% (v/v), less than about 15% (v/v) or less thanabout 20% (v/v). In some embodiments, PDX1-positive foregut endodermcells are grown without serum. In other embodiments, PDX1-positiveforegut endoderm cells are grown with serum replacement.

In still other embodiments, PDX1-positive foregut endoderm cells aregrown in the presence of B27. In such embodiments, B27 can be providedto the culture medium in concentrations ranging from about 0.1% (v/v) toabout 20% (v/v) or in concentrations greater than about 20% (v/v). Incertain embodiments, the concentration of B27 in the medium is about0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4%(v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v),about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20% (v/v).Alternatively, the concentration of the added B27 supplement can bemeasured in terms of multiples of the strength of a commerciallyavailable B27 stock solution. For example, B27 is available fromInvitrogen (Carlsbad, Calif.) as a 50× stock solution. Addition of asufficient amount of this stock solution to a sufficient volume ofgrowth medium produces a medium supplemented with the desired amount ofB27. For example, the addition of 10 ml of 50×B27 stock solution to 90ml of growth medium would produce a growth medium supplemented with5×B27. The concentration of B27 supplement in the medium can be about0.1×, about 0.2×, about 0.3×, about 0.4×, about 0.5×, about 0.6×, about0.7×, about 0.8×, about 0.9×, about 1×, about 1.1×, about 1.2×, about1.3×, about 1.4×, about 1.5×, about 1.6×, about 1.7×, about 1.8×, about1.9×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×,about 5×, about 6×, about 7×, about 8×, about 9×, about 10×, about 11×,about 12×, about 13×, about 14×, about 15×, about 16×, about 17×, about18×, about 19×, about 20× and greater than about 20×.

Production of Dorsal PDX1-Positive Foregut Endoderm from PDX1-NegativeDefinitive Endoderm

The dorsal PDX1-positive foregut endoderm cell cultures and populationscomprising dorsal PDX1-positive foregut endoderm cells that aredescribed herein are produced from PDX1-negative definitive endoderm,which is generated from pluripotent cells as described above.Furthermore, as described above, a preferred method utilizes humanembryonic stem cells as the starting material. In one embodiment, hESCsare first converted to PDX1-negative definitive endoderm cells, whichare then converted to dorsal PDX1-positive foregut endoderm cells. Itwill be appreciated, however, that the starting materials for theproduction of dorsal PDX1-positive foregut endoderm is not limited todefinitive endoderm cells produced using pluripotent celldifferentiation methods. Rather, any PDX1-negative definitive endodermcells can be used in the methods described herein regardless of theirorigin.

As described in connection with the production of mixed populations ofPDX1-positive foregut endoderm cells, in some embodiments of the presentinvention, cell cultures or cell populations comprising PDX1-negativedefinitive endoderm cells can be used for further differentiation tocell cultures and/or enriched cell populations comprising dorsalPDX1-positive foregut endoderm cells. For example, a cell culture orcell population comprising human PDX1-negative, SOX17-positivedefinitive endoderm cells can be used. In some embodiments, the cellculture or cell population may also comprise differentiation factors,such as activins, nodals and/or BMPs, remaining from the previousdifferentiation step (that is, the step of differentiating pluripotentcells to definitive endoderm cells). In other embodiments, factorsutilized in the previous differentiation step are removed from the cellculture or cell population prior to the addition of factors used for thedifferentiation of the PDX1-negative, SOX17-positive definitive endodermcells to dorsal PDX1-positive foregut endoderm cells. In otherembodiments, cell populations enriched for PDX1-negative, SOX17-positivedefinitive endoderm cells are used as a source for the production ofdorsal PDX1-positive foregut endoderm cells.

PDX1-negative definitive endoderm cells in culture are differentiated todorsal PDX1-positive endoderm cells by providing to a cell culturecomprising PDX1-negative, SOX17-positive definitive endoderm cells aretinoid, such as retinoic acid (RA). In some embodiments, the retinoidis used in conjunction with a member of the TGFβ superfamily of growthfactors and/or Connaught Medical Research Labs medium (CRML medium)(Invitrogen, Carlsbad, Calif.).

With respect to some of the embodiments of differentiation processesdescribed herein, the RA or a combination of the above-mentioneddifferentiation factors are provided to the cells so that these factorsare present in the cell culture or cell population at concentrationssufficient to promote differentiation of at least a portion of thePDX1-negative definitive endoderm cell culture or cell population todorsal PDX1-positive foregut endoderm cells. When used in connectionwith cell cultures and/or cell populations, the term “portion” means anynon-zero amount of the cell culture or cell population, which rangesfrom a single cell to the entirety of the cell culture or cellpopulation. In preferred embodiments, the term “portion” means at least5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19%, at least 20%, atleast 21%, at least 22%, at least 23%, at least 24%, at least 25%, atleast 26%, at least 27%, at least 28%, at least 29%, at least 30%, atleast 31%, at least 32%, at least 33%, at least 34%, at least 35%, atleast 36%, at least 37%, at least 38%, at least 39%, at least 40%, atleast 41%, at least 42%, at least 43%, at least 44%, at least 45%, atleast 46%, at least 47%, at least 48%, at least 49%, at least 50%, atleast 51%, at least 52%, at least 53%, at least 54%, at least 55%, atleast 56%, at least 57%, at least 58%, at least 59%, at least 60%, atleast 61%, at least 62%, at least 63%, at least 64%, at least 65%, atleast 66%, at least 67%, at least 68%, at least 69%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74% or at least 75% ofthe cell culture or cell population.

In some embodiments of the present invention, a retinoid is provided tothe cells of a cell culture such that it is present at a concentrationof at least about 0.01 μM, at least about 0.02 μM, at least about 0.04μM, at least about 0.08 μM, at least about 0.1 μM, at least about 0.2μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM,at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, atleast about 0.9 μM, at least about 1 μM, at least about 1.1 μM, at leastabout 1.2 μM, at least about 1.3 μM, at least about 1.4 μM, at leastabout 1.5 μM, at least about 1.6 μM, at least about 1.7 μM, at leastabout 1.8 μM, at least about 1.9 μM, at least about 2 μM, at least about2.1 μM, at least about 2.2 μM, at least about 2.3 μM, at least about 2.4μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM,at least about 2.8 μM, at least about 2.9 μM, at least about 3 μM, atleast about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at leastabout 5 μM, at least about 10 μM, at least about 20 μM, at least about30 μM, at least about 40 μM or at least about 50 μM.

In preferred embodiments of the present invention, a population ofdorsally-biased PDX1-positive foregut endoderm cells is produced byproviding retinoic acid in the absence of exogenous FGF-10 or other FGFfamily growth factor. In such embodiments, RA is provided at aconcentration of about 2 μM. In a preferred embodiment, RA is providedat a concentration of about 2 μM in CMRL medium.

In some embodiments, activin A and/or activin B are provided to the cellculture along with RA. For example, in some embodiments, RA is providedto the cell culture at a concentration of about 2 μM and activin Aand/or activin B is provided to the cell culture at a concentration ofat least about 5 ng/ml, at least about 10 ng/ml, at least about 25ng/ml, at least about 50 ng/ml, at least about 75 ng/ml, at least about100 ng/ml, at least about 200 ng/ml, at least about 300 ng/ml, at leastabout 400 ng/ml, at least about 500 ng/ml, or at least about 1000 ng/ml.

In some embodiments, the differentiation factors and/or CRML medium isprovided to the PDX1-negative definitive endoderm cells at about threedays, at about four days, at about five days, at about six days, atabout seven days, at about eight days, at about nine days, at about tendays or at about greater than ten days subsequent to the initiation ofdifferentiation from hESCs. In preferred embodiments, differentiationfactors and/or CRML medium is provided to the PDX1-negative definitiveendoderm cells at about five days subsequent to the initiation ofdifferentiation from hESCs.

In certain embodiments of the present invention, the above-mentioneddifferentiation factors are removed from the cell culture subsequent totheir addition. For example, the above-mentioned differentiation factorscan be removed within about one day, about two days, about three days,about four days, about five days, about six days, about seven days,about eight days, about nine days or about ten days after theiraddition.

Cultures of dorsal PDX1-positive foregut endoderm cells can be grown ina medium containing reduced serum. Serum concentrations can range fromabout 0.05% (v/v) to about 20% (v/v). In some embodiments, dorsalPDX1-positive foregut endoderm cells are grown with serum replacement.For example, in certain embodiments, the serum concentration of themedium can be less than about 0.05% (v/v), less than about 0.1% (v/v),less than about 0.2% (v/v), less than about 0.3% (v/v), less than about0.4% (v/v), less than about 0.5% (v/v), less than about 0.6% (v/v), lessthan about 0.7% (v/v), less than about 0.8% (v/v), less than about 0.9%(v/v), less than about 1% (v/v), less than about 2% (v/v), less thanabout 3% (v/v), less than about 4% (v/v), less than about 5% (v/v), lessthan about 6% (v/v), less than about 7% (v/v), less than about 8% (v/v),less than about 9% (v/v), less than about 10% (v/v), less than about 15%(v/v) or less than about 20% (v/v). In some embodiments, dorsalPDX1-positive foregut endoderm cells are grown without serum. In otherembodiments, dorsal PDX1-positive foregut endoderm cells are grown withserum replacement.

In still other embodiments, dorsal PDX1-positive foregut endoderm cellsare grown in the presence of B27. In such embodiments, B27 can beprovided to the culture medium in concentrations ranging from about 0.1%(v/v) to about 20% (v/v) or in concentrations greater than about 20%(v/v). In certain embodiments, the concentration of B27 in the medium isabout 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v),about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v),about 0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about4% (v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8%(v/v), about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20%(v/v). Alternatively, the concentration of the added B27 supplement canbe measured in terms of multiples of the strength of a commerciallyavailable B27 stock solution. For example, B27 is available fromInvitrogen (Carlsbad, Calif.) as a 50× stock solution. Addition of asufficient amount of this stock solution to a sufficient volume ofgrowth medium produces a medium supplemented with the desired amount ofB27. For example, the addition of 10 ml of 50×B27 stock solution to 90ml of growth medium would produce a growth medium supplemented with5×B27. The concentration of B27 supplement in the medium can be about0.1×, about 0.2×, about 0.3×, about 0.4×, about 0.5×, about 0.6×, about0.7×, about 0.8×, about 0.9×, about 1×, about 1.1×, about 1.2×, about1.3×, about 1.4×, about 1.5×, about 1.6×, about 1.7×, about 1.8×, about1.9×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×,about 5×, about 6×, about 7×, about 8×, about 9×, about 10×, about 11×,about 12×, about 13×, about 14×, about 15×, about 16×, about 17×, about18×, about 19×, about 20× and greater than about 20×.

Production of Ventral PDX1-Positive Foregut Endoderm from PDX1-NegativeDefinitive Endoderm

The ventral PDX1-positive foregut endoderm cell cultures and populationscomprising ventral PDX1-positive foregut endoderm cells that aredescribed herein are produced from PDX1-negative definitive endoderm,which is generated from pluripotent cells as described above.Furthermore, as described above, a preferred method utilizes humanembryonic stem cells as the starting material. In one embodiment, hESCsare first converted to PDX1-negative definitive endoderm cells, whichare then converted to ventral PDX1-positive foregut endoderm cells. Itwill be appreciated, however, that the starting materials for theproduction of ventral PDX1-positive foregut endoderm is not limited todefinitive endoderm cells produced using pluripotent celldifferentiation methods. Rather, any PDX1-negative definitive endodermcells can be used in the methods described herein regardless of theirorigin.

As described in connection with the production of mixed populations ofPDX1-positive foregut endoderm cells, in some embodiments of the presentinvention, cell cultures or cell populations comprising PDX1-negativedefinitive endoderm cells can be used for further differentiation tocell cultures and/or enriched cell populations comprising ventralPDX1-positive foregut endoderm cells. For example, a cell culture orcell population comprising human PDX1-negative, SOX17-positivedefinitive endoderm cells can be used. In some embodiments, the cellculture or cell population may also comprise differentiation factors,such as activins, nodals and/or BMPs, remaining from the previousdifferentiation step (that is, the step of differentiating pluripotentcells to definitive endoderm cells). In other embodiments, factorsutilized in the previous differentiation step are removed from the cellculture or cell population prior to the addition of factors used for thedifferentiation of the PDX1-negative, SOX17-positive definitive endodermcells to ventral PDX1-positive foregut endoderm cells. In otherembodiments, cell populations enriched for PDX1-negative, SOX17-positivedefinitive endoderm cells are used as a source for the production ofventral PDX1-positive foregut endoderm cells.

PDX1-negative definitive endoderm cells in culture are differentiated toventral PDX1-positive endoderm cells by providing to a cell culturecomprising PDX1-negative, SOX17-positive definitive endoderm cells anFGF-family growth factor or FGF-family growth factor analog or mimetic.In some embodiments, the FGF-family growth factor or FGF-family growthfactor analog or mimetic is used in conjunction with a hedgehoginhibitor and/or Connaught Medical Research Labs medium (CRML medium)(Invitrogen, Carlsbad, Calif.). In especially preferred embodiments,FGF-10 and/or KAAD-cyclopamine is provided to a cell culture comprisingPDX1-negative definitive endoderm cells in the absence of RA or otherretinoid. In certain embodiments, BMP4 may be included in FGF-10 and/orKAAD-cyclopamine in the absence of RA or other retinoid. After about oneday to about ten days subsequent to the addition of the FGF-familygrowth factor analog or mimetic and/or hedgehog inhibitor, a retinoid,such as RA, or a retinoid containing supplement, such as B27, isprovided to induce the expression of PDX1. In a preferred embodiments,RA is provided at about 2 days, about 3 day, about 4 day or about 5 dayssubsequent to the addition of the FGF-family growth factor analog ormimetic and/or hedgehog inhibitor. In other embodiments, B27 is providedat about the same time as providing the FGF-family growth factor analogor mimetic and/or hedgehog inhibitor.

With respect to some of the embodiments of differentiation processesdescribed herein, a retinoid and a combination of the above-mentioneddifferentiation factors are provided to the cells so that these factorsare present in the cell culture or cell population at concentrationssufficient to promote differentiation of at least a portion of thePDX1-negative definitive endoderm cell culture or cell population toventral PDX1-positive foregut endoderm cells. When used in connectionwith cell cultures and/or cell populations, the term “portion” means anynon-zero amount of the cell culture or cell population, which rangesfrom a single cell to the entirety of the cell culture or cellpopulation. In preferred embodiments, the term “portion” means at least5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19%, at least 20%, atleast 21%, at least 22%, at least 23%, at least 24%, at least 25%, atleast 26%, at least 27%, at least 28%, at least 29%, at least 30%, atleast 31%, at least 32%, at least 33%, at least 34%, at least 35%, atleast 36%, at least 37%, at least 38%, at least 39%, at least 40%, atleast 41%, at least 42%, at least 43%, at least 44%, at least 45%, atleast 46%, at least 47%, at least 48%, at least 49%, at least 50%, atleast 51%, at least 52%, at least 53%, at least 54%, at least 55%, atleast 56%, at least 57%, at least 58%, at least 59%, at least 60%, atleast 61%, at least 62%, at least 63%, at least 64%, at least 65%, atleast 66%, at least 67%, at least 68%, at least 69%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74% or at least 75% ofthe cell culture or cell population.

In some embodiments of the present invention, the FGF-family growthfactor or FGF-family growth factor analog or mimetic is provided to thecells of a cell culture such that it is present at a concentration of atleast about 10 ng/ml, at least about 25 ng/ml, at least about 50 ng/ml,at least about 75 ng/ml, at least about 100 ng/ml, at least about 200ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at leastabout 500 ng/ml, or at least about 1000 ng/ml. In other embodiments,when used alone or in conjunction with FGF-10, KAAD-cyclopamine can beprovided at a concentration of at least about 0.01 μM, at least about0.02 μM, at least about 0.04 μM, at least about 0.08 μM, at least about0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4μM, at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM,at least about 0.8 μM, at least about 0.9 μM, at least about 1 μM, atleast about 1.1 μM, at least about 1.2 μM, at least about 1.3 μM, atleast about 1.4 μM, at least about 1.5 μM, at least about 1.6 μM, atleast about 1.7 μM, at least about 1.8 μM, at least about 1.9 μM, atleast about 2 μM, at least about 2.1 μM, at least about 2.2 μM, at leastabout 2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at leastabout 2.6 μM, at least about 2.7 μM, at least about 2.8 μM, at leastabout 2.9 μM, at least about 3 μM, at least about 3.5 μM, at least about4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM,at least about 20 μM, at least about 30 μM, at least about 40 μM or atleast about 50 μM.

In preferred embodiments of the present invention, a population ofventrally-biased PDX1-positive foregut endoderm cells is produced byproviding a population of PDX1-negative definitive endoderm with 50ng/ml of FGF-10 and 0.5 μM KAAD-cyclopamine in CMRL medium in theabsence of RA. About two days subsequent to the addition of FGF-10 andKAAD-cyclopamine 2 μM RA is added to complete the differentiation of thecells to PDX1-positive cells.

In some embodiments, the differentiation factors and/or CRML medium isprovided to the PDX1-negative definitive endoderm cells at about threedays, at about four days, at about five days, at about six days, atabout seven days, at about eight days, at about nine days, at about tendays or at about greater than ten days subsequent to the initiation ofdifferentiation from hESCs. In preferred embodiments, differentiationfactors and/or CRML medium is provided to the PDX1-negative definitiveendoderm cells at about three days subsequent to the initiation ofdifferentiation from hESCs.

In certain embodiments of the present invention, the above-mentioneddifferentiation factors are removed from the cell culture subsequent totheir addition. For example, the above-mentioned differentiation factorscan be removed within about one day, about two days, about three days,about four days, about five days, about six days, about seven days,about eight days, about nine days or about ten days after theiraddition.

Cultures of ventral PDX1-positive foregut endoderm cells can be grown ina medium containing reduced serum. Serum concentrations can range fromabout 0.05% (v/v) to about 20% (v/v). In some embodiments, ventralPDX1-positive foregut endoderm cells are grown with serum replacement.For example, in certain embodiments, the serum concentration of themedium can be less than about 0.05% (v/v), less than about 0.1% (v/v),less than about 0.2% (v/v), less than about 0.3% (v/v), less than about0.4% (v/v), less than about 0.5% (v/v), less than about 0.6% (v/v), lessthan about 0.7% (v/v), less than about 0.8% (v/v), less than about 0.9%(v/v), less than about 1% (v/v), less than about 2% (v/v), less thanabout 3% (v/v), less than about 4% (v/v), less than about 5% (v/v), lessthan about 6% (v/v), less than about 7% (v/v), less than about 8% (v/v),less than about 9% (v/v), less than about 10% (v/v), less than about 15%(v/v) or less than about 20% (v/v). In some embodiments, ventralPDX1-positive foregut endoderm cells are grown without serum. In otherembodiments, ventral PDX1-positive foregut endoderm cells are grown withserum replacement.

In still other embodiments, ventral PDX1-positive foregut endoderm cellsare grown in the presence of B27. In such embodiments, B27 can beprovided to the culture medium in concentrations ranging from about 0.1%(v/v) to about 20% (v/v) or in concentrations greater than about 20%(v/v). In certain embodiments, the concentration of B27 in the medium isabout 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v),about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v),about 0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about4% (v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8%(v/v), about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20%(v/v). Alternatively, the concentration of the added B27 supplement canbe measured in terms of multiples of the strength of a commerciallyavailable B27 stock solution. For example, B27 is available fromInvitrogen (Carlsbad, Calif.) as a 50× stock solution. Addition of asufficient amount of this stock solution to a sufficient volume ofgrowth medium produces a medium supplemented with the desired amount ofB27. For example, the addition of 10 ml of 50×B27 stock solution to 90ml of growth medium would produce a growth medium supplemented with5×B27. The concentration of B27 supplement in the medium can be about0.1×, about 0.2×, about 0.3×, about 0.4×, about 0.5×, about 0.6×, about0.7×, about 0.8×, about 0.9×, about 1×, about 1.1×, about 1.2×, about1.3×, about 1.4×, about 1.5×, about 1.6×, about 1.7×, about 1.8×, about1.9×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×,about 5×, about 6×, about 7×, about 8×, about 9×, about 10×, about 11×,about 12×, about 13×, about 14×, about 15×, about 16×, about 17×, about18×, about 19×, about 20× and greater than about 20×. In someembodiments where B27 is provided, a retinoid is not provided tocomplete the differentiation of the PDX1-negative cells to ventralPDX1-positive foregut endoderm.

Monitoring the Differentiation of PDX1-Negative Definitive Endoderm toPDX1-Positive Foregut Endoderm

As with the differentiation of definitive endoderm cells frompluripotent cells, the progression of differentiation fromPDX1-negative, SOX17-positive definitive endoderm to PDX1-positiveforegut endoderm can be monitored by determining the expression ofmarkers characteristic of these cell types. Such monitoring permits oneto determine the amount of time that is sufficient for the production ofa desired amount of PDX1-positive foregut endoderm under variousconditions, for example, one or more differentiation factorconcentrations and environmental conditions. In preferred embodiments,the amount of time that is sufficient for the production of a desiredamount of PDX1-positive foregut endoderm is determined by detecting theexpression of PDX1. In some embodiments of the present invention, theexpression of certain markers is determined by detecting the presence orabsence of the marker. Alternatively, the expression of certain markerscan be determined by measuring the level at which the marker is presentin the cells of the cell culture or cell population. In suchembodiments, the measurement of marker expression can be qualitative orquantitative. As described above, a preferred method of quantitating theexpression markers that are produced by marker genes is through the useof Q-PCR. In particular embodiments, Q-PCR is used to monitor theprogression of cells of the PDX1-negative, SOX17-positive definitiveendoderm culture to PDX1-positive foregut endoderm cells by quantitatingexpression of marker genes characteristic of PDX1-positive foregutendoderm and the lack of expression of marker genes characteristic ofother cell types. Other methods which are known in the art can also beused to quantitate marker gene expression. For example, the expressionof a marker gene product can be detected by using antibodies specificfor the marker gene product of interest. In some embodiments of thepresent invention, the expression of marker genes characteristic ofPDX1-positive foregut endoderm as well as the lack of significantexpression of marker genes characteristic of PDX1-negative definitiveendoderm, hESCs and other cell types is determined.

As described further in the Examples below, PDX1 is a marker gene thatis associated with PDX1-positive foregut endoderm. As such, in someembodiments of the present invention, the expression of PDX1 isdetermined. In other embodiments, the expression of other markers, whichare expressed in PDX1-positive foregut endoderm, including, but notlimited to, SOX17, HOXA13 and/or HOXC6 is also determined. Since PDX1can also be expressed by certain other cell types (that is, visceralendoderm and certain neural ectoderm), some embodiments of the presentinvention relate to demonstrating the absence or substantial absence ofmarker gene expression that is associated with visceral endoderm and/orneural ectoderm. For example, in some embodiments, the expression ofmarkers, which are expressed in visceral endoderm and/or neural cells,including, but not limited to, SOX7, AFP, SOX1, ZIC1 and/or NFM isdetermined

In some embodiments, PDX1-positive foregut endoderm cell culturesproduced by the methods described herein are substantially free of cellsexpressing the SOX7, AFP, SOX1, ZIC1 or NFM marker genes. In certainembodiments, the PDX1-positive foregut endoderm cell cultures producedby the processes described herein are substantially free of visceralendoderm, parietal endoderm and/or neural cells.

Monitoring the Differentiation of PDX1-Negative Definitive Endoderm toDorsal PDX1-Positive Foregut Endoderm

Expression of one or more of the markers described in Table 3 and/orTable 4, in the Examples below, can be detected and/or quantitated usingthe above-described methods, such as Q-PCR and/or immunocytochemistry,to monitor the differentiation of PDX1-negative definitive endoderm todorsal PDX1-positive endoderm. Markers associated with bothdorsally-biased and ventrally-biased PDX1-positive foregut endodermcells are described in Table 3. Of these markers, the markers selectedfrom the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3,LGALS3/GALIG, SERPINF2 and SLC27A2 are cell surface markers. Somepreferred markers listed in Table 3 for monitoring the production ofdorsal PDX1-positive foregut endoderm are selected from the groupconsisting of SERPINF2, DUSP9, CDH6 and SOX9. Markers associated withdorsally-biased foregut endoderm are described in Table 4. Each of theTable 4 markers is expressed preferentially, specifically or uniquely indorsal PDX1-positive foregut endoderm cells as compared to otherPDX1-positive cells. Of these markers, the markers selected from thegroup consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10,SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4 and XPR1 are cell surfacemarkers. Some preferred markers listed in Table 4 for monitoring theproduction of dorsal PDX1-positive foregut endoderm are selected fromthe group consisting of HOXA1, PDE11A, FAM49A and WNT5A.

In addition to the above-described markers, in some embodiments of thepresent invention, the expression of other markers, which are expressedin PDX1-positive foregut endoderm, including, but not limited to, SOX17,HOXA13 and/or HOXC6 is also determined. Since PDX1 can also be expressedby certain other cell types (that is, visceral endoderm and certainneural ectoderm), some embodiments of the present invention relate todemonstrating the absence or substantial absence of marker geneexpression that is associated with visceral endoderm and/or neuralectoderm. For example, in some embodiments, the expression of markers,which are expressed in visceral endoderm and/or neural cells, including,but not limited to, SOX7, AFP, SOX1, ZIC1 and/or NFM is determined

In some embodiments, dorsal PDX1-positive foregut endoderm cell culturesproduced by the methods described herein are substantially free of cellsexpressing the SOX7, AFP, SOX1, ZIC1 or NFM marker genes. In certainembodiments, the dorsal PDX1-positive foregut endoderm cell culturesproduced by the processes described herein are substantially free ofvisceral endoderm, parietal endoderm and/or neural cells.

Monitoring the Differentiation of PDX1-Negative Definitive Endoderm toVentral PDX1-Positive Foregut Endoderm

As described in the previous section, markers associated with bothdorsally-biased and ventrally-biased PDX1-positive foregut endodermcells are described in Table 3. As such, expression of one or more ofthe markers described in Table 3 can be detected and/or quantitatedusing the above-described methods, such as Q-PCR and/orimmunocytochemistry, to monitor the differentiation of PDX1-negativedefinitive endoderm to ventral PDX1-positive endoderm. Of the markersdescribed in Table 3, the markers selected from the group consisting ofCDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 andSLC27A2 are cell surface markers. Some preferred markers listed in Table3 for monitoring the production of ventral PDX1-positive foregutendoderm are selected from the group consisting of SERPINF2, DUSP9, CDH6and SOX9. Furthermore, because the markers described in Table 4 arepreferentially, specifically or uniquely expressed in dorsally-biasedforegut endoderm, detecting the lack of expression, or reducedexpression relative to the expression in dorsal PDX1-positive foregutendoderm, of one or more of these markers is also useful for monitoringthe differentiation of PDX1-negative definitive endoderm to ventralPDX1-positive foregut endoderm. Of the Table 4 markers, the markersselected from the group consisting of ADORA2A, CD47, EPB41L1, MAG,SFRP5, SLC16A10, SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4 and XPR1 arecell surface markers. Some preferred markers listed in Table 4 formonitoring the production of dorsal PDX1-positive foregut endoderm areselected from the group consisting of HOXA1, PDE11A, FAM49A and WNT5A.As such, the absence, or insubstantial expression, of these markers inPDX1-positive cells expressing one or more markers selected from Table3, is indicative of ventral PDX1-positive

In addition to the above-described markers, in some embodiments of thepresent invention, the expression of other markers, which are expressedin PDX1-positive foregut endoderm, including, but not limited to, SOX17,HOXA13 and/or HOXC6 is also determined. Since PDX1 can also be expressedby certain other cell types (that is, visceral endoderm and certainneural ectoderm), some embodiments of the present invention relate todemonstrating the absence or substantial absence of marker geneexpression that is associated with visceral endoderm and/or neuralectoderm. For example, in some embodiments, the expression of markers,which are expressed in visceral endoderm and/or neural cells, including,but not limited to, SOX7, AFP, SOX1, ZIC1 and/or NFM is determined

In some embodiments, ventral PDX1-positive foregut endoderm cellcultures produced by the methods described herein are substantially freeof cells expressing the SOX7, AFP, SOX1, ZIC1 or NFM marker genes. Incertain embodiments, the ventral PDX1-positive foregut endoderm cellcultures produced by the processes described herein are substantiallyfree of visceral endoderm, parietal endoderm and/or neural cells.

Enrichment, Isolation and/or Purification of Dorsal and/or VentralPDX1-Positive Foregut Endoderm

PDX1-positive foregut endoderm cells, including dorsal and/or ventralPDX1-positive foregut endoderm cells, produced by any of theabove-described processes can be enriched, isolated and/or purified byusing an affinity tag that is specific for such cells. Examples ofaffinity tags specific for dorsal and/or ventral PDX1-positive foregutendoderm cells are antibodies, ligands or other binding agents that arespecific to a marker molecule, such as a polypeptide, that is present onthe cell surface of dorsal and/or ventral PDX1-positive foregut endodermcells but which is not substantially present on other cell types thatwould be found in a cell culture produced by the methods describedherein. In some processes, an antibody which binds to a cell surfacemarker selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R,KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, ADORA2A, CD47, EPB41L1,MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4 and XPR1is used as an affinity tag for the enrichment, isolation or purificationof dorsal and/or ventral PDX1-positive foregut endoderm cells.

Methods for making antibodies and using them for cell isolation areknown in the art and such methods can be implemented for use with theantibodies and dorsal and/or ventral PDX1-positive foregut endodermcells described herein. In one process, an antibody which binds to amarker selected from CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3,LGALS3/GALIG, SERPINF2, SLC27A2, ADORA2A, CD47, EPB41L1, MAG, SFRP5,SLC16A10, SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4 and XPR1 is attachedto a magnetic bead and then allowed to bind to dorsal and/or ventralPDX1-positive foregut endoderm cells in a cell culture which has beenenzymatically treated to reduce intercellular and substrate adhesion.The cell/antibody/bead complexes are then exposed to a movable magneticfield which is used to separate bead-bound definitive endoderm cellsfrom unbound cells. Once the dorsal and/or ventral PDX1-positive foregutendoderm cells are physically separated from other cells in culture, theantibody binding is disrupted and the cells are replated in appropriatetissue culture medium.

Additional methods for obtaining enriched, isolated or purified dorsaland/or ventral PDX1-positive foregut endoderm cell cultures orpopulations can also be used. For example, in some embodiments, anantibody that binds to a marker selected from the group consisting ofCDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2,SLC27A2, ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3,SLC30A4, SLICK, SLITRK4 and XPR1 is incubated with a dorsal and/orventral PDX1-positive foregut endoderm-containing cell culture that hasbeen treated to reduce intercellular and substrate adhesion. The cellsare then washed, centrifuged and resuspended. The cell suspension isthen incubated with a secondary antibody, such as an FITC-conjugatedantibody that is capable of binding to the primary antibody. The cellsare then washed, centrifuged and resuspended in buffer. The cellsuspension is then analyzed and sorted using a fluorescence activatedcell sorter (FACS). The marker-positive cells are collected separatelyfrom marker-negative cells, thereby resulting in the isolation of suchcell types. If desired, the isolated cell compositions can be furtherpurified by using an alternate affinity-based method or by additionalrounds of sorting using the same or different markers that are specificfor dorsal and/or ventral PDX1-positive foregut endoderm cells.

In still other processes, dorsal and/or ventral PDX1-positive foregutendoderm cells are enriched, isolated and/or purified using a ligand orother molecule that binds to a marker selected from the group consistingof CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2,SLC27A2, ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3,SLC30A4, SLICK, SLITRK4 and XPR1.

In preferred processes, dorsal and/or ventral PDX1-positive foregutendoderm cells are enriched, isolated and/or purified from other cellsafter the PDX1-negative definitive endoderm cell cultures are induced todifferentiate towards the dorsal and/or ventral PDX1-positive foregutendoderm lineage. It will be appreciated that the above-describedenrichment, isolation and purification procedures can be used with suchcultures at any stage of differentiation.

In addition to the procedures just described, dorsal and/or ventralPDX1-positive foregut endoderm cells may also be isolated by othertechniques for cell isolation. Additionally, dorsal and/or ventralPDX1-positive foregut endoderm cells may also be enriched or isolated bymethods of serial subculture in growth conditions which promote theselective survival or selective expansion of the dorsal and/or ventralPDX1-positive foregut endoderm cells.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of dorsal and/or ventral PDX1-positive foregut endodermcells and or tissues can be produced in vitro from PDX1-negativedefinitive endoderm cell cultures or cell populations, which haveundergone at least some differentiation. In some methods, the cellsundergo random differentiation. In a preferred method, however, thecells are directed to differentiate primarily into dorsal and/or ventralPDX1-positive foregut endoderm cells. Some preferred enrichment,isolation and/or purification methods relate to the in vitro productionof dorsal and/or ventral PDX1-positive foregut endoderm cells from humanPDX1-negative definitive endoderm cells. Using the methods describedherein, cell populations or cell cultures can be enriched in dorsaland/or ventral PDX1-positive foregut endoderm content by at least about2- to about 1000-fold as compared to untreated cell populations or cellcultures. In some embodiments, dorsal and/or ventral PDX1-positiveforegut endoderm cells can be enriched by at least about 5- to about500-fold as compared to untreated cell populations or cell cultures. Inother embodiments, dorsal and/or ventral PDX1-positive foregut endodermcells can be enriched from at least about 10- to about 200-fold ascompared to untreated cell populations or cell cultures. In still otherembodiments, dorsal and/or ventral PDX1-positive foregut endoderm cellscan be enriched from at least about 20- to about 100-fold as compared tountreated cell populations or cell cultures. In yet other embodiments,dorsal and/or ventral PDX1-positive foregut endoderm cells can beenriched from at least about 40- to about 80-fold as compared tountreated cell populations or cell cultures. In certain embodiments,dorsal and/or ventral PDX1-positive foregut endoderm cells can beenriched from at least about 2- to about 20-fold as compared tountreated cell populations or cell cultures.

Enrichment, Isolation and/or Purification of Dorsal and/or VentralPDX1-Positive Foregut Endoderm

With respect to additional aspects of the present invention, dorsaland/or ventral PDX1-positive foregut endoderm cells can be enriched,isolated and/or purified. In some embodiments of the present invention,cell populations enriched for dorsal and/or ventral PDX1-positiveforegut endoderm cells are produced by isolating such cells from cellcultures.

In some embodiments of the present invention, dorsal and/or ventralPDX1-positive foregut endoderm cells are fluorescently labeled thenisolated from non-labeled cells by using a fluorescence activated cellsorter (FACS). In such embodiments, a nucleic acid encoding fluorescentprotein (GFP) or another nucleic acid encoding an expressiblefluorescent marker gene, such as the gene encoding luciferase, is usedto label PDX1-positive cells. For example, in some embodiments, at leastone copy of a nucleic acid encoding GFP or a biologically activefragment thereof is introduced into a pluripotent cell, preferably ahuman embryonic stem cell, downstream of the promoter of a gene selectedfrom Table 3 or Table 4 such that the expression of the GFP gene productor biologically active fragment thereof is under control of the suchpromoter. In some embodiments, the entire coding region of the nucleicacid, which encodes the marker selected from Table 3 or Table 4, isreplaced by a nucleic acid encoding GFP or a biologically activefragment thereof. In other embodiments, the nucleic acid encoding GFP ora biologically active fragment thereof is fused in frame with at least aportion of the nucleic acid encoding the marker selected from Table 3 orTable 4, thereby generating a fusion protein. In such embodiments, thefusion protein retains a fluorescent activity similar to GFP.

Fluorescently marked cells, such as the above-described pluripotentcells, are differentiated to definitive endoderm and then to dorsaland/or ventral PDX1-positive foregut endoderm cells as describedpreviously above. Because dorsal and/or ventral PDX1-positive foregutendoderm cells express the fluorescent marker gene, whereasPDX1-negative cells do not, these two cell types can be separated. Insome embodiments, cell suspensions comprising a mixture offluorescently-labeled dorsal and/or ventral PDX1-positive foregutendoderm cells and unlabeled PDX1-negative cells are sorted using aFACS. Dorsal and/or ventral PDX1-positive foregut endoderm cells arecollected separately from PDX1-negative cells, thereby resulting in theisolation of such cell types. If desired, the isolated cell compositionscan be further purified by additional rounds of sorting using the sameor different markers that are specific for dorsal and/or ventralPDX1-positive foregut endoderm.

It will be appreciated that the above-described enrichment, isolationand purification procedures can be used with such cultures at any stageof differentiation.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of dorsal and/or ventral PDX1-positive foregut endodermcells and/or tissues can be produced in vitro from PDX1-negative,SOX17-positive definitive endoderm cell cultures or cell populationswhich have undergone at least some differentiation. In some embodiments,the cells undergo random differentiation. In a preferred embodiment,however, the cells are directed to differentiate primarily into dorsaland/or ventral PDX1-positive foregut endoderm cells. Some preferredenrichment, isolation and/or purification methods relate to the in vitroproduction of dorsal and/or ventral PDX1-positive foregut endoderm cellsfrom human embryonic stem cells.

Using the methods described herein, cell populations or cell culturescan be enriched in dorsal and/or ventral PDX1-positive foregut endodermcell content by at least about 2- to about 1000-fold as compared tountreated cell populations or cell cultures. In some embodiments, dorsaland/or ventral PDX1-positive foregut endoderm cells can be enriched byat least about 5- to about 500-fold as compared to untreated cellpopulations or cell cultures. In other embodiments, dorsal and/orventral PDX1-positive foregut endoderm cells can be enriched from atleast about 10- to about 200-fold as compared to untreated cellpopulations or cell cultures. In still other embodiments, dorsal and/orventral PDX1-positive foregut endoderm cells can be enriched from atleast about 20- to about 100-fold as compared to untreated cellpopulations or cell cultures. In yet other embodiments, dorsal and/orventral PDX1-positive foregut endoderm cells can be enriched from atleast about 40- to about 80-fold as compared to untreated cellpopulations or cell cultures. In certain embodiments, dorsal and/orventral PDX1-positive foregut endoderm cells can be enriched from atleast about 2- to about 20-fold as compared to untreated cellpopulations or cell cultures.

Compositions Comprising Dorsal and/or Ventral PDX1-Positive ForegutEndoderm

Some embodiments of the present invention relate to cell compositions,such as cell cultures or cell populations, comprising dorsal and/orventral PDX1-positive foregut endoderm cells, wherein the dorsal and/orventral PDX1-positive foregut endoderm cells are multipotent cells thatcan differentiate into cells, tissues or organs derived from theanterior portion of the gut tube, such as the dorsal pancreatic budand/or the ventral pancreatic bud. In accordance with certainembodiments, the dorsal and/or ventral PDX1-positive foregut endodermcells are mammalian cells, and in a preferred embodiment, such cells arehuman cells.

Other embodiments of the present invention relate to compositions, suchas cell cultures or cell populations, comprising cells of one or morecell types selected from the group consisting of hESCs, PDX1-negativedefinitive endoderm cells, dorsal and/or ventral PDX1-positive foregutendoderm cells and mesoderm cells. In some embodiments, hESCs compriseless than about 5%, less than about 4%, less than about 3%, less thanabout 2% or less than about 1% of the total cells in the culture. Inother embodiments, PDX1-negative definitive endoderm cells comprise lessthan about 90%, less than about 85%, less than about 80%, less thanabout 75%, less than about 70%, less than about 65%, less than about60%, less than about 55%, less than about 50%, less than about 45%, lessthan about 40%, less than about 35%, less than about 30%, less thanabout 25%, less than about 20%, less than about 15%, less than about12%, less than about 10%, less than about 8%, less than about 6%, lessthan about 5%, less than about 4%, less than about 3%, less than about2% or less than about 1% of the total cells in the culture. In yet otherembodiments, mesoderm cells comprise less than about 90%, less thanabout 85%, less than about 80%, less than about 75%, less than about70%, less than about 65%, less than about 60%, less than about 55%, lessthan about 50%, less than about 45%, less than about 40%, less thanabout 35%, less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 12%, less than about 10%, lessthan about 8%, less than about 6%, less than about 5%, less than about4%, less than about 3%, less than about 2% or less than about 1% of thetotal cells in the culture.

Additional embodiments of the present invention relate to compositions,such as cell cultures or cell populations, produced by the processesdescribed herein comprise dorsal and/or ventral PDX1-positive foregutendoderm as the majority cell type. In some embodiments, the processesdescribed herein produce cell cultures and/or cell populationscomprising at least about 99%, at least about 98%, at least about 97%,at least about 96%, at least about 95%, at least about 94%, at leastabout 93%, at least about 92%, at least about 91%, at least about 90%,at least about 89%, at least about 88%, at least about 87%, at leastabout 86%, at least about 85%, at least about 84%, at least about 83%,at least about 82%, at least about 81%, at least about 80%, at leastabout 79%, at least about 78%, at least about 77%, at least about 76%,at least about 75%, at least about 74%, at least about 73%, at leastabout 72%, at least about 71%, at least about 70%, at least about 69%,at least about 68%, at least about 67%, at least about 66%, at leastabout 65%, at least about 64%, at least about 63%, at least about 62%,at least about 61%, at least about 60%, at least about 59%, at leastabout 58%, at least about 57%, at least about 56%, at least about 55%,at least about 54%, at least about 53%, at least about 52%, at leastabout 51% or at least about 50% dorsal and/or ventral PDX1-positiveforegut endoderm cells. In preferred embodiments the cells of the cellcultures or cell populations comprise human cells. In other embodiments,the processes described herein produce cell cultures or cell populationscomprising at least about 50%, at least about 45%, at least about 40%,at least about 35%, at least about 30%, at least about 25%, at leastabout 24%, at least about 23%, at least about 22%, at least about 21%,at least about 20%, at least about 19%, at least about 18%, at leastabout 17%, at least about 16%, at least about 15%, at least about 14%,at least about 13%, at least about 12%, at least about 11%, at leastabout 10%, at least about 9%, at least about 8%, at least about 7%, atleast about 6%, at least about 5%, at least about 4%, at least about 3%,at least about 2% or at least about 1% dorsal and/or ventralPDX1-positive foregut endoderm cells. In preferred embodiments, thecells of the cell cultures or cell populations comprise human cells. Insome embodiments, the percentage of dorsal and/or ventral PDX1-positiveforegut endoderm cells in the cell cultures or populations is calculatedwithout regard to the feeder cells remaining in the culture.

Still other embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising mixtures of dorsaland/or ventral PDX1-positive foregut endoderm cells and PDX1-negativedefinitive endoderm cells. For example, cell cultures or cellpopulations comprising at least about 5 dorsal and/or ventralPDX1-positive foregut endoderm cells for about every 95 PDX1-negativedefinitive endoderm cells can be produced. In other embodiments, cellcultures or cell populations comprising at least about 95 dorsal and/orventral PDX1-positive foregut endoderm cells for about every 5PDX1-negative definitive endoderm cells can be produced. Additionally,cell cultures or cell populations comprising other ratios of dorsaland/or ventral PDX1-positive foregut endoderm cells to PDX1-negativedefinitive endoderm cells are contemplated. For example, compositionscomprising at least about 1 dorsal or ventral PDX1-positive foregutendoderm cell for about every 1,000,000 PDX1-negative definitiveendoderm cells, at least about 1 dorsal or ventral PDX1-positive foregutendoderm cell for about every 100,000 PDX1-negative definitive endodermcells, at least about 1 dorsal or ventral PDX1-positive foregut endodermcell for about every 10,000 PDX1-negative definitive endoderm cells, atleast about 1 dorsal or ventral PDX1-positive foregut endoderm cell forabout every 1000 PDX1-negative definitive endoderm cells, at least about1 dorsal or ventral PDX1-positive foregut endoderm cell for about every500 PDX1-negative definitive endoderm cells, at least about 1 dorsal orventral PDX1-positive foregut endoderm cell for about every 100PDX1-negative definitive endoderm cells, at least about 1 dorsal orventral PDX1-positive foregut endoderm cell for about every 10PDX1-negative definitive endoderm cells, at least about 1 dorsal orventral PDX1-positive foregut endoderm cell for about every 5PDX1-negative definitive endoderm cells, at least about 1 dorsal orventral PDX1-positive foregut endoderm cell for about every 4PDX1-negative definitive endoderm cells, at least about 1 dorsal orventral PDX1-positive foregut endoderm cell for about every 2PDX1-negative definitive endoderm cells, at least about 1 dorsal orventral PDX1-positive foregut endoderm cell for about every 1PDX1-negative definitive endoderm cell, at least about 2 dorsal and/orventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 4 dorsal and/orventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 5 dorsal and/orventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 10 dorsal and/orventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 20 dorsal and/orventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 50 dorsal and/orventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 100 dorsal and/orventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 1000 dorsaland/or ventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 10,000 dorsaland/or ventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 100,000 dorsaland/or ventral PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell and at least about 1,000,000dorsal and/or ventral PDX1-positive foregut endoderm cells for aboutevery 1 PDX1-negative definitive endoderm cell are contemplated.

In some embodiments of the present invention, the PDX1-negativedefinitive endoderm cells from which dorsal and/or ventral PDX1-positiveforegut endoderm cells are produced are derived from human pluripotentcells, such as human pluripotent stem cells. In certain embodiments, thehuman pluripotent cells are derived from a morula, the inner cell massof an embryo or the gonadal ridges of an embryo. In certain otherembodiments, the human pluripotent cells are derived from the gonadal orgerm tissues of a multicellular structure that has developed past theembryonic stage.

Further embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising human cells,including human dorsal and/or ventral PDX1-positive foregut endodermcells, wherein the expression of the PDX1 marker is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 2% of the human cells. In other embodiments, the expression of thePDX1 marker is greater than the expression of the AFP, SOX7, SOX1, ZIC1and/or NFM marker in at least about 5% of the human cells, in at leastabout 10% of the human cells, in at least about 15% of the human cells,in at least about 20% of the human cells, in at least about 25% of thehuman cells, in at least about 30% of the human cells, in at least about35% of the human cells, in at least about 40% of the human cells, in atleast about 45% of the human cells, in at least about 50% of the humancells, in at least about 55% of the human cells, in at least about 60%of the human cells, in at least about 65% of the human cells, in atleast about 70% of the human cells, in at least about 75% of the humancells, in at least about 80% of the human cells, in at least about 85%of the human cells, in at least about 90% of the human cells, in atleast about 95% of the human cells or in at least about 98% of the humancells. In some embodiments, the percentage of human cells in the cellcultures or populations, wherein the expression of PDX1 is greater thanthe expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker, iscalculated without regard to feeder cells.

It will be appreciated that some embodiments of the present inventionrelate to compositions, such as cell cultures or cell populations,comprising human dorsal and/or ventral PDX1-positive foregut endodermcells, wherein the expression of one or more markers selected from thegroup consisting of SOX17, HOXA13 and HOXC6 is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in from atleast about 2% to greater than at least about 98% of the human cells. Insome embodiments, the expression of one or more markers selected fromthe group consisting of SOX17, HOXA13 and HOXC6 is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 5% of the human cells, in at least about 10% of the human cells,in at least about 15% of the human cells, in at least about 20% of thehuman cells, in at least about 25% of the human cells, in at least about30% of the human cells, in at least about 35% of the human cells, in atleast about 40% of the human cells, in at least about 45% of the humancells, in at least about 50% of the human cells, in at least about 55%of the human cells, in at least about 60% of the human cells, in atleast about 65% of the human cells, in at least about 70% of the humancells, in at least about 75% of the human cells, in at least about 80%of the human cells, in at least about 85% of the human cells, in atleast about 90% of the human cells, in at least about 95% of the humancells or in at least about 98% of the human cells. In some embodiments,the percentage of human cells in the cell cultures or populations,wherein the expression of one or more markers selected from the groupconsisting of SOX17, HOXA13 and HOXC6 is greater than the expression ofthe AFP, SOX7, SOX1, ZIC1 and/or NFM marker, is calculated withoutregard to feeder cells.

Other embodiments of the present invention relate to compositions, suchas cell cultures or cell populations, comprising human cells, includinghuman dorsal and/or ventral PDX1-positive foregut endoderm cells,wherein the expression of one or more markers selected from Table 3 isgreater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFMmarker in at least about 2% of the human cells. In other embodiments,the expression of the one or more markers selected from Table 3 isgreater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFMmarker in at least about 5% of the human cells, in at least about 10% ofthe human cells, in at least about 15% of the human cells, in at leastabout 20% of the human cells, in at least about 25% of the human cells,in at least about 30% of the human cells, in at least about 35% of thehuman cells, in at least about 40% of the human cells, in at least about45% of the human cells, in at least about 50% of the human cells, in atleast about 55% of the human cells, in at least about 60% of the humancells, in at least about 65% of the human cells, in at least about 70%of the human cells, in at least about 75% of the human cells, in atleast about 80% of the human cells, in at least about 85% of the humancells, in at least about 90% of the human cells, in at least about 95%of the human cells or in at least about 98% of the human cells. In someembodiments, the percentage of human cells in the cell cultures orpopulations, wherein the expression of one or more markers selected fromTable 3 is greater than the expression of the AFP, SOX7, SOX1, ZIC1and/or NFM marker, is calculated without regard to feeder cells.

Other embodiments of the present invention relate to compositions, suchas cell cultures or cell populations, comprising human cells, includinghuman dorsal PDX1-positive foregut endoderm cells, wherein theexpression of one or more markers selected from Table 4 is greater thanthe expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in atleast about 2% of the human cells. In other embodiments, the expressionof the one or more markers selected from Table 4 is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 5% of the human cells, in at least about 10% of the human cells,in at least about 15% of the human cells, in at least about 20% of thehuman cells, in at least about 25% of the human cells, in at least about30% of the human cells, in at least about 35% of the human cells, in atleast about 40% of the human cells, in at least about 45% of the humancells, in at least about 50% of the human cells, in at least about 55%of the human cells, in at least about 60% of the human cells, in atleast about 65% of the human cells, in at least about 70% of the humancells, in at least about 75% of the human cells, in at least about 80%of the human cells, in at least about 85% of the human cells, in atleast about 90% of the human cells, in at least about 95% of the humancells or in at least about 98% of the human cells. In some embodiments,the percentage of human cells in the cell cultures or populations,wherein the expression of one or more markers selected from Table 4 isgreater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFMmarker, is calculated without regard to feeder cells.

Other embodiments of the present invention relate to compositions, suchas cell cultures or cell populations, comprising human cells, includinghuman ventral PDX1-positive foregut endoderm cells, wherein theexpression of a marker selected from Table 3 is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 2% of the human cells, and wherein a marker selected from Table 4is not substantially expressed as compared to the expression of the samemarker in dorsal PDX1-positive foregut endoderm cells. In otherembodiments, the expression of the marker selected from Table 3 isgreater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFMmarker in at least about 5% of the human cells, in at least about 10% ofthe human cells, in at least about 15% of the human cells, in at leastabout 20% of the human cells, in at least about 25% of the human cells,in at least about 30% of the human cells, in at least about 35% of thehuman cells, in at least about 40% of the human cells, in at least about45% of the human cells, in at least about 50% of the human cells, in atleast about 55% of the human cells, in at least about 60% of the humancells, in at least about 65% of the human cells, in at least about 70%of the human cells, in at least about 75% of the human cells, in atleast about 80% of the human cells, in at least about 85% of the humancells, in at least about 90% of the human cells, in at least about 95%of the human cells or in at least about 98% of the human cells. In suchembodiments, a marker selected from Table 4 is not substantiallyexpressed as compared to the expression of the same marker in dorsalPDX1-positive foregut endoderm cells. In some embodiments, thepercentage of human cells in the cell cultures or populations, whereinthe expression of a marker selected from Table 3 is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker, and wherein amarker selected from Table 4 is not substantially expressed as comparedto the expression of the same marker in dorsal PDX1-positive foregutendoderm cells, is calculated without regard to feeder cells.

Additional embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising mammalianendodermal cells, such as human endoderm cells, wherein the expressionof the PDX1 marker and the expression of one or more markers selectedfrom Table 3 or Table 4 is greater than the expression of the AFP, SOX7,SOX1, ZIC1 and/or NFM marker in at least about 2% of the endodermalcells. In other embodiments, the expression of the PDX1 marker and theexpression of one or more markers selected from Table 3 or Table 4 isgreater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFMmarker in at least about 5% of the endodermal cells, in at least about10% of the endodermal cells, in at least about 15% of the endodermalcells, in at least about 20% of the endodermal cells, in at least about25% of the endodermal cells, in at least about 30% of the endodermalcells, in at least about 35% of the endodermal cells, in at least about40% of the endodermal cells, in at least about 45% of the endodermalcells, in at least about 50% of the endodermal cells, in at least about55% of the endodermal cells, in at least about 60% of the endodermalcells, in at least about 65% of the endodermal cells, in at least about70% of the endodermal cells, in at least about 75% of the endodermalcells, in at least about 80% of the endodermal cells, in at least about85% of the endodermal cells, in at least about 90% of the endodermalcells, in at least about 95% of the endodermal cells or in at leastabout 98% of the endodermal cells.

Still other embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising mammalianendodermal cells, such as human endodermal cells, wherein the expressionof one or more markers selected from the group consisting of SOX17,HOXA13 and HOXC6 and the expression of one or more markers selected fromTable 3 or Table 4 is greater than the expression of the AFP, SOX7,SOX1, ZIC1 and/or NFM marker in at least about 2% of the endodermalcells. In other embodiments, the expression of one or more markersselected from the group consisting of SOX17, HOXA13 and HOXC6 and theexpression of one or more markers selected from Table 3 or Table 4 isgreater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFMmarker in at least about 5% of the endodermal cells, in at least about10% of the endodermal cells, in at least about 15% of the endodermalcells, in at least about 20% of the endodermal cells, in at least about25% of the endodermal cells, in at least about 30% of the endodermalcells, in at least about 35% of the endodermal cells, in at least about40% of the endodermal cells, in at least about 45% of the endodermalcells, in at least about 50% of the endodermal cells, in at least about55% of the endodermal cells, in at least about 60% of the endodermalcells, in at least about 65% of the endodermal cells, in at least about70% of the endodermal cells, in at least about 75% of the endodermalcells, in at least about 80% of the endodermal cells, in at least about85% of the endodermal cells, in at least about 90% of the endodermalcells, in at least about 95% of the endodermal cells or at least about98% of the endodermal cells.

Using the processes described herein, compositions comprising dorsaland/or ventral PDX1-positive foregut endoderm cells substantially freeof other cell types can be produced. With respect to cells in cellcultures or in cell populations, the term “substantially free of” meansthat the specified cell type of which the cell culture or cellpopulation is free, is present in an amount of less than about 5% of thetotal number of cells present in the cell culture or cell population. Insome embodiments of the present invention, the dorsal and/or ventralPDX1-positive foregut endoderm cell populations or cell culturesproduced by the methods described herein are substantially free of cellsthat significantly express the AFP, SOX7, SOX1, ZIC1 and/or NFM markergenes.

In one embodiment of the present invention, a description of a dorsalPDX1-positive foregut endoderm cell based on the expression of markergenes is, PDX1 high, a marker selected from Table 3 high, a markerselected from Table 4 high, AFP low, SOX7 low, SOX1 low, ZIC1 low andNFM low.

In one embodiment of the present invention, a description of a ventralPDX1-positive foregut endoderm cell based on the expression of markergenes is, PDX1 high, a marker selected from Table 3 high, a markerselected from Table 4 low as compared to the expression of the samemarker in dorsal PDX1-positive foregut endoderm, AFP low, SOX7 low, SOX1low, ZIC1 low and NFM low.

Increasing Expression of PDX1 in a SOX17-Positive Definitive EndodermCell

Some aspects of the present invention are related to methods ofincreasing the expression of the PDX1 gene product in cell cultures orcell populations comprising SOX17-positive definitive endoderm cells. Insuch embodiments, the SOX17-positive definitive endoderm cells arecontacted with a differentiation factor in an amount that is sufficientto increase the expression of the PDX1 gene product. The SOX17-positivedefinitive endoderm cells that are contacted with the differentiationfactor can be either PDX1-negative or PDX1-positive. In someembodiments, the differentiation factor can be a retinoid. In certainembodiments, SOX17-positive definitive endoderm cells are contacted witha retinoid at a concentration ranging from about 0.01 μM to about 50 μM.In a preferred embodiment, the retinoid is RA.

In other embodiments of the present invention, the expression of thePDX1 gene product in cell cultures or cell populations comprisingSOX17-positive definitive endoderm cells is increased by contacting theSOX17-positive cells with a differentiation factor of the fibroblastgrowth factor family. Such differentiation factors can either be usedalone or in conjunction with RA. In some embodiments, the SOX17-positivedefinitive endoderm cells are contacted with a fibroblast growth factorat a concentration ranging from about 10 ng/ml to about 1000 ng/ml. In apreferred embodiment, the FGF growth factor is FGF-10.

In some embodiments of the present invention, the expression of the PDX1gene product in cell cultures or cell populations comprisingSOX17-positive definitive endoderm cells is increased by contacting theSOX17-positive cells with B27. This differentiation factor can either beused alone or in conjunction with one or both of retinoid and FGF familydifferentiation factors. In some embodiments, the SOX17-positivedefinitive endoderm cells are contacted with B27 at a concentrationranging from about 0.1% (v/v) to about 20% (v/v). In a preferredembodiment, the SOX17-positive definitive endoderm cells are contactedwith RA, FGF-10 and B27.

Methods for increasing the expression of the PDX1 gene product in cellcultures or cell populations comprising SOX17-positive definitiveendoderm cells can be carried out in growth medium containing reduced orno serum. In some embodiments, serum concentrations range from about0.05% (v/v) to about 20% (v/v). In some embodiments, the SOX17-positivecells are grown with serum replacement.

It will be appreciated that the above described methods can also be usedto increase the expression of one or more markers selected from Table 3and/or Table 4 in dorsal PDX1-positive foregut endoderm cells.Similarly, such methods can be used to increase the expression of one ormore markers selected from Table 3 in ventral PDX1-positive foregutendoderm cells.

Identification of Factors Capable of Promoting the Differentiation ofPDX1-Negative Definitive Endoderm Cells to PDX1-Positive ForegutEndoderm Cells

Additional aspects of the present invention relate to methods ofidentifying one or more differentiation factors capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. In such methods, a cell culture orcell population comprising PDX1-negative definitive endoderm cells isobtained and the expression of PDX1 in the cell culture or cellpopulation is determined. After determining the expression of PDX1, thecells of the cell culture or cell population are contacted with acandidate differentiation factor. In some embodiments, the expression ofPDX1 is determined at the time of contacting or shortly after contactingthe cells with a candidate differentiation factor. PDX1 expression isthen determined at one or more times after contacting the cells with thecandidate differentiation factor. If the expression of PDX1 hasincreased after contact with the candidate differentiation factor ascompared to PDX1 expression prior to contact with the candidatedifferentiation factor, the candidate differentiation factor isidentified as capable of promoting the differentiation of PDX1-negativedefinitive endoderm cells to PDX1-positive foregut endoderm cells.

In some embodiments, the above-described methods of identifying factorscapable of promoting the differentiation of PDX1-negative definitiveendoderm cells to PDX1-positive foregut endoderm cells also includedetermining the expression of the HOXA13 gene and/or the HOXC6 gene inthe cell culture or cell population. In such embodiments, the expressionof HOXA13 and/or HOXC6 is determined both before and after the cells arecontacted with the candidate differentiation factor. If the expressionof PDX1 and HOXA13 has increased after contact with the candidatedifferentiation factor as compared to PDX1 and HOXA13 expression priorto contact with the candidate differentiation factor, the candidatedifferentiation factor is identified as capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. Similarly, if the expression ofPDX1 and HOXC6 has increased after contact with the candidatedifferentiation factor as compared to PDX1 and HOXC6 expression prior tocontact with the candidate differentiation factor, the candidatedifferentiation factor is identified as capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. In a preferred embodiment, acandidate differentiation factor is identified as being capable ofpromoting the differentiation of PDX1-negative definitive endoderm cellsto PDX1-positive foregut endoderm cells by determining the expression ofPDX1, HOXA13 and HOXC6 both before and after contacting the cells of thecell culture or cell population with the candidate differentiationfactor. In preferred embodiments, the expression of PDX1, HOXA13 and/orHOXC6 is determined Q-PCR.

It will be appreciated that in some embodiments, the expression of oneor more of PDX1, HOXA13 and HOXC6 can be determined at the time ofcontacting or shortly after contacting the cells of the cell cultures orcell populations with a candidate differentiation factor rather thanprior to contacting the cells with a candidate differentiation factor.In such embodiments, the expression of one or more of PDX1, HOXA13 andHOXC6 at the time of contacting or shortly after contacting the cellswith a candidate differentiation factor is compared to the expression ofone or more of PDX1, HOXA13 and HOXC6 at one or more times aftercontacting the cells with a candidate differentiation factor.

In some embodiments of the above-described methods, the one or moretimes at which PDX1 expression is determined after contacting the cellswith the candidate differentiation factor can range from about 1 hour toabout 10 days. For example, PDX1 expression can be determined about 1hour after contacting the cells with the candidate differentiationfactor, about 2 hours after contacting the cells with the candidatedifferentiation factor, about 4 hours after contacting the cells withthe candidate differentiation factor, about 6 hours after contacting thecells with the candidate differentiation factor, about 8 hours aftercontacting the cells with the candidate differentiation factor, about 10hours after contacting the cells with the candidate differentiationfactor, about 12 hours after contacting the cells with the candidatedifferentiation factor, about 16 hours after contacting the cells withthe candidate differentiation factor, about 24 hours after contactingthe cells with the candidate differentiation factor, about 2 days aftercontacting the cells with the candidate differentiation factor, about 3days after contacting the cells with the candidate differentiationfactor, about 4 days after contacting the cells with the candidatedifferentiation factor, about 5 days after contacting the cells with thecandidate differentiation factor, about 6 days after contacting thecells with the candidate differentiation factor, about 7 days aftercontacting the cells with the candidate differentiation factor, about 8days after contacting the cells with the candidate differentiationfactor, about 9 days after contacting the cells with the candidatedifferentiation factor, about 10 days after contacting the cells withthe candidate differentiation factor or more than 10 days aftercontacting the cells with the candidate differentiation factor.

Candidate differentiation factors for use in the methods describedherein can be selected from compounds, such as polypeptides and smallmolecules. For example, candidate polypeptides can include, but are notlimited to, growth factors, cytokines, chemokines, extracellular matrixproteins, and synthetic peptides. In a preferred embodiment, the growthfactor is from the FGF family, for example FGF-10. Candidate smallmolecules include, but are not limited to, compounds generated fromcombinatorial chemical synthesis and natural products, such as steroids,isoprenoids, terpenoids, phenylpropanoids, alkaloids and flavinoids. Itwill be appreciated by those of ordinary skill in the art that thousandsof classes of natural and synthetic small molecules are available andthat the small molecules contemplated for use in the methods describedherein are not limited to the classes exemplified above. Typically,small molecules will have a molecular weight less than 10,000 amu. In apreferred embodiment, the small molecule is a retinoid, for example RA.

It will be appreciated that the above-described methods can be used toidentify factors capable of promoting the differentiation ofPDX1-negative definitive endoderm cells to dorsal PDX1-positive foregutendoderm cells by monitoring the expression of one or more markersexpression from Table 4. In some embodiments, the expression of one ormore markers selected from Table 3 and one or more markers selected fromTable 4 is monitored.

Similarly, it will also be appreciated that the above-described methodscan be used to identify factors capable of promoting the differentiationof PDX1-negative definitive endoderm cells to ventral PDX1-positiveforegut endoderm cells by monitoring the expression of one or moremarkers expression from Table 3. In some embodiments, the expression ofone or more markers selected from Table 3 and one or more markersselected from Table 4 is monitored.

Identification of Factors Capable of Promoting the Differentiation ofDorsal and/or Ventral PDX1-Positive Foregut Endoderm Cells

Certain screening methods described herein relate to methods foridentifying at least one differentiation factor that is capable ofpromoting the differentiation of dorsal and/or ventral PDX1-positiveforegut endoderm cells. In some embodiments of these methods, cellpopulations comprising dorsal and/or ventral PDX1-positive foregutendoderm cells, such as human dorsal and/or ventral PDX1-positiveforegut endoderm cells, are obtained. The cell population is thenprovided with a candidate differentiation factor. At a first time point,which is prior to or at approximately the same time as providing thecandidate differentiation factor, expression of a marker is determined.Alternatively, expression of the marker can be determined afterproviding the candidate differentiation factor. At a second time point,which is subsequent to the first time point and subsequent to the stepof providing the candidate differentiation factor to the cellpopulation, expression of the same marker is again determined. Whetherthe candidate differentiation factor is capable of promoting thedifferentiation of the dorsal and/or ventral PDX1-positive foregutendoderm cells is determined by comparing expression of the marker atthe first time point with the expression of the marker at the secondtime point. If expression of the marker at the second time point isincreased or decreased as compared to expression of the marker at thefirst time point, then the candidate differentiation factor is capableof promoting the differentiation of dorsal and/or ventral PDX1-positiveforegut endoderm cells. In preferred embodiments, expression of themarker is determined by Q-PCR.

Some embodiments of the screening methods described herein utilize cellpopulations or cell cultures which comprise human dorsal and/or ventralPDX1-positive foregut endoderm cells. For example, the cell populationcan be a substantially purified population of human dorsal and/orventral PDX1-positive foregut endoderm cells. Alternatively, the cellpopulation can be an enriched population of human dorsal and/or ventralPDX1-positive foregut endoderm cells, wherein at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97% orgreater than at least about 97% of the human cells in the cellpopulation are human dorsal and/or ventral PDX1-positive foregutendoderm cells. In other embodiments described herein, the cellpopulation comprises human cells wherein at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85% or greater than at least about 85% of the human cells arehuman dorsal and/or ventral PDX1-positive foregut endoderm cells. Insome embodiments, the cell population includes non-human cells such asnon-human feeder cells. In other embodiments, the cell populationincludes human feeder cells. In such embodiments, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95% or greater thanat least about 95% of the human cells, other than said feeder cells, arehuman dorsal and/or ventral PDX1-positive foregut endoderm cells.

In embodiments of the screening methods described herein, the cellpopulation is contacted or otherwise provided with a candidate (test)differentiation factor. The candidate differentiation factor cancomprise any molecule that may have the potential to promote thedifferentiation of human dorsal and/or ventral PDX1-positive foregutendoderm cells. In some embodiments described herein, the candidatedifferentiation factor comprises a molecule that is known to be adifferentiation factor for one or more types of cells. In alternateembodiments, the candidate differentiation factor comprises a moleculethat in not known to promote cell differentiation. In preferredembodiments, the candidate differentiation factor comprises moleculethat is not known to promote the differentiation of dorsal and/orventral PDX1-positive foregut endoderm cells.

In some embodiments of the screening methods described herein, thecandidate differentiation factor comprises a small molecule. Inpreferred embodiments, a small molecule is a molecule having a molecularmass of about 10,000 amu or less. In some embodiments, the smallmolecule comprises a retinoid. In some embodiments, the small moleculecomprises retinoic acid.

In other embodiments described herein, the candidate differentiationfactor comprises a polypeptide. The polypeptide can be any polypeptideincluding, but not limited to, a glycoprotein, a lipoprotein, anextracellular matrix protein, a cytokine, a chemokine, a peptidehormone, an interleukin or a growth factor. Preferred polypeptidesinclude growth factors.

In some embodiments of the screening methods described herein, thecandidate differentiation factors comprise one or more growth factorsselected from the group consisting of Amphiregulin, B-lymphocytestimulator, IL-16, Thymopoietin, TRAIL/Apo-2, Pre B cell colonyenhancing factor, Endothelial differentiation-related factor 1 (EDF1),Endothelial monocyte activating polypeptide II, Macrophage migrationinhibitory factor (MIF), Natural killer cell enhancing factor (NKEFA),Bone mophogenetic protein 2, Bone mophogenetic protein 8 (osteogeneicprotein 2), Bone morphogenic protein 6, Bone morphogenic protein 7,Connective tissue growth factor (CTGF), CGI-149 protein (neuroendocrinedifferentiation factor), Cytokine A3 (macrophage inflammatory protein1-alpha), Gliablastoma cell differentiation-related protein (GBDR1),Hepatoma-derived growth factor, Neuromedin U-25 precursor, Vascularendothelial growth factor (VEGF), Vascular endothelial growth factor B(VEGF-B), T-cell specific RANTES precursor, thymic dendriticcell-derived factor 1, Transferrin, Interleukin-1 (IL 1), Interleukin-2(IL 2), Interleukin-3 (IL 3), Interleukin-4 (IL 4), Interleukin-5 (IL5), Interleukin-6 (IL 6), Interleukin-7 (IL 7), Interleukin-8 (IL 8),Interleukin-9 (IL 9), Interleukin-10 (IL 10), Interleukin-11 (IL 11),Interleukin-12 (IL 12), Interleukin-13 (IL 13), Granulocyte-colonystimulating factor (G-CSF), Granulocyte macrophage colony stimulatingfactor (GM-CSF), Macrophage colony stimulating factor (M-CSF),Erythropoietin, Thrombopoietin, Vitamin D₃, Epidermal growth factor(EGF), Brain-derived neurotrophic factor, Leukemia inhibitory factor,Thyroid hormone, Basic fibroblast growth factor (bFGF), aFGF, FGF-4,FGF-6, Keratinocyte growth factor (KGF), Platelet-derived growth factor(PDGF), Platelet-derived growth factor-BB, beta nerve growth factor,activin A, Transforming growth factor beta 1 (TGF-β1), Interferon-α,Interferon-β, Interferon-γ, Tumor necrosis factor-α, Tumor necrosisfactor-β, Burst promoting activity (BPA), Erythroid promoting activity(EPA), PGE₂, insulin growth factor-1 (IGF-1), IGF-II, Neutrophin growthfactor (NGF), Neutrophin-3, Neutrophin 4/5, Ciliary neurotrophic factor,Glial-derived nexin, Dexamethasone, β-mercaptoethanol, Retinoic acid,Butylated hydroxyanisole, 5-azacytidine, Amphotericin B, Ascorbic acid,Ascrorbate, isobutylxanthine, indomethacin, β-glycerolphosphate,nicotinamide, DMSO, Thiazolidinediones, TWS119, oxytocin, vasopressin,melanocyte-stimulating hormone, corticortropin, lipotropin, thyrotropin,growth hormone, prolactin, luteinizing hormone, human chorionicgonadotropin, follicle stimulating hormone, corticotropin-releasingfactor, gonadotropin-releasing factor, prolactin-releasing factor,prolactin-inhibiting factor, growth-hormone releasing factor,somatostatin, thyrotropin-releasing factor, calcitonin gene-relatedpeptide, parathyroid hormone, glucagon-like peptide 1, glucose-dependentinsulinotropic polypeptide, gastrin, secretin, cholecystokinin, motilin,vasoactive intestinal peptide, substance P, pancreatic polypeptide,peptide tyrosine tyrosine, neuropeptide tyrosine, insulin, glucagon,placental lactogen, relaxin, angiotensin II, calctriol, atrialnatriuretic peptide, and melatonin, thyroxine, triiodothyronine,calcitonin, estradiol, estrone, progesterone, testosterone, cortisol,corticosterone, aldosterone, epinephrine, norepinepherine, androstiene,calcitriol, collagen, Dexamethasone, β-mercaptoethanol, Retinoic acid,Butylated hydroxyanisole, 5-azacytidine, Amphotericin B, Ascorbic acid,Ascrorbate, isobutylxanthine, indomethacin, β-glycerolphosphate,nicotinamide, DMSO, Thiazolidinediones, and TWS119.

In some embodiments of the screening methods described herein, thecandidate differentiation factor is provided to the cell population inone or more concentrations. In some embodiments, the candidatedifferentiation factor is provided to the cell population so that theconcentration of the candidate differentiation factor in the mediumsurrounding the cells ranges from about 0.1 ng/ml to about 10 mg/ml. Insome embodiments, the concentration of the candidate differentiationfactor in the medium surrounding the cells ranges from about 1 ng/ml toabout 1 mg/ml. In other embodiments, the concentration of the candidatedifferentiation factor in the medium surrounding the cells ranges fromabout 10 ng/ml to about 100 μg/ml. In still other embodiments, theconcentration of the candidate differentiation factor in the mediumsurrounding the cells ranges from about 100 ng/ml to about 10 μg/ml. Inpreferred embodiments, the concentration of the candidatedifferentiation factor in the medium surrounding the cells is about 5ng/ml, about 25 ng/ml, about 50 ng/ml, about 75 ng/ml, about 100 ng/ml,about 125 ng/ml, about 150 ng/ml, about 175 ng/ml, about 200 ng/ml,about 225 ng/ml, about 250 ng/ml, about 275 ng/ml, about 300 ng/ml,about 325 ng/ml, about 350 ng/ml, about 375 ng/ml, about 400 ng/ml,about 425 ng/ml, about 450 ng/ml, about 475 ng/ml, about 500 ng/ml,about 525 ng/ml, about 550 ng/ml, about 575 ng/ml, about 600 ng/ml,about 625 ng/ml, about 650 ng/ml, about 675 ng/ml, about 700 ng/ml,about 725 ng/ml, about 750 ng/ml, about 775 ng/ml, about 800 ng/ml,about 825 ng/ml, about 850 ng/ml, about 875 ng/ml, about 900 ng/ml,about 925 ng/ml, about 950 ng/ml, about 975 ng/ml, about 1 μg/ml, about2 μg/ml, about 3 μg/ml, about 4 μg/ml, about 5 μg/ml, about 6 μg/ml,about 7 μg/ml, about 8 μg/ml, about 9 μg/ml, about 10 μg/ml, about 11μg/ml, about 12 μg/ml, about 13 μg/ml, about 14 μg/ml, about 15 μg/ml,about 16 μg/ml, about 17 μg/ml, about 18 μg/ml, about 19 μg/ml, about 20μg/ml, about 25 μg/ml, about 50 μg/ml, about 75 μg/ml, about 100 μg/ml,about 125 μg/ml, about 150 μg/ml, about 175 μg/ml, about 200 μg/ml,about 250 μg/ml, about 300 μg/ml, about 350 μg/ml, about 400 μg/ml,about 450 μg/ml, about 500 μg/ml, about 550 μg/ml, about 600 μg/ml,about 650 μg/ml, about 700 μg/ml, about 750 μg/ml, about 800 μg/ml,about 850 μg/ml, about 900 μg/ml, about 950 μg/ml, about 1000 μg/ml orgreater than about 1000 μg/ml.

In certain embodiments of the screening methods described herein, thecell population is provided with a candidate differentiation factorwhich comprises any molecule other than a retinoid, FGF-10, FGF-4,BMP-4, activin A, activin B or any other foregut differentiation factor.In some embodiments, the cell population is provided with a candidatedifferentiation factor which comprises any molecule other than retinoicacid.

In some embodiments, steps of the screening methods described hereincomprise determining expression of at least one marker at a first timepoint and a second time point. In some of these embodiments, the firsttime point can be prior to or at approximately the same time asproviding the cell population with the candidate differentiation factor.Alternatively, in some embodiments, the first time point is subsequentto providing the cell population with the candidate differentiationfactor. In some embodiments, expression of a plurality of markers isdetermined at a first time point.

In addition to determining expression of at least one marker at a firsttime point, some embodiments of the screening methods described hereincontemplate determining expression of at least one marker at a secondtime point, which is subsequent to the first time point and which issubsequent to providing the cell population with the candidatedifferentiation factor. In such embodiments, expression of the samemarker is determined at both the first and second time points. In someembodiments, expression of a plurality of markers is determined at boththe first and second time points. In such embodiments, expression of thesame plurality of markers is determined at both the first and secondtime points. In some embodiments, marker expression is determined at aplurality of time points, each of which is subsequent to the first timepoint, and each of which is subsequent to providing the cell populationwith the candidate differentiation factor. In certain embodiments,marker expression is determined by Q-PCR. In other embodiments, markerexpression is determined by immunocytochemistry.

In certain embodiments of the screening methods described herein, themarker having its expression is determined at the first and second timepoints is a marker that is associated with the differentiation of humandorsal and/or ventral PDX1-positive foregut endoderm cells to cellswhich are the precursors of cells which make up tissues and/or organsthat are derived from the posterior portion of the foregut. In someembodiments, the tissues and/or organs that are derived from theposterior portion of the foregut comprise terminally differentiatedcells. In some embodiments, the marker is indicative of pancreatic cellsor pancreatic precursor cells. In some embodiments, the marker is amarker that is selected from Table 3 or Table 4.

In some embodiments of the screening methods described herein,sufficient time is allowed to pass between providing the cell populationwith the candidate differentiation factor and determining markerexpression at the second time point. Sufficient time between providingthe cell population with the candidate differentiation factor anddetermining expression of the marker at the second time point can be aslittle as from about 1 hour to as much as about 10 days. In someembodiments, the expression of at least one marker is determinedmultiple times subsequent to providing the cell population with thecandidate differentiation factor. In some embodiments, sufficient timeis at least about 1 hour, at least about 6 hours, at least about 12hours, at least about 18 hours, at least about 24 hours, at least about30 hours, at least about 36 hours, at least about 42 hours, at leastabout 48 hours, at least about 54 hours, at least about 60 hours, atleast about 66 hours, at least about 72 hours, at least about 78 hours,at least about 84 hours, at least about 90 hours, at least about 96hours, at least about 102 hours, at least about 108 hours, at leastabout 114 hours, at least about 120 hours, at least about 126 hours, atleast about 132 hours, at least about 138 hours, at least about 144hours, at least about 150 hours, at least about 156 hours, at leastabout 162 hours, at least about 168 hours, at least about 174 hours, atleast about 180 hours, at least about 186 hours, at least about 192hours, at least about 198 hours, at least about 204 hours, at leastabout 210 hours, at least about 216 hours, at least about 222 hours, atleast about 228 hours, at least about 234 hours or at least about 240hours.

In some embodiments of the methods described herein, it is furtherdetermined whether the expression of the marker at the second time pointhas increased or decreased as compared to the expression of this markerat the first time point. An increase or decrease in the expression ofthe at least one marker indicates that the candidate differentiationfactor is capable of promoting the differentiation of the dorsal and/orventral PDX1-positive foregut endoderm cells. Similarly, if expressionof a plurality of markers is determined, it is further determinedwhether the expression of the plurality of markers at the second timepoint has increased or decreased as compared to the expression of thisplurality of markers at the first time point. An increase or decrease inmarker expression can be determined by measuring or otherwise evaluatingthe amount, level or activity of the marker in the cell population atthe first and second time points. Such determination can be relative toother markers, for example housekeeping gene expression, or absolute. Incertain embodiments, wherein marker expression is increased at thesecond time point as compared with the first time point, the amount ofincrease is at least about 2-fold, at least about 5-fold, at least about10-fold, at least about 20-fold, at least about 30-fold, at least about40-fold, at least about 50-fold, at least about 60-fold, at least about70-fold, at least about 80-fold, at least about 90-fold, at least about100-fold or more than at least about 100-fold. In some embodiments, theamount of increase is less than 2-fold. In embodiments where markerexpression is decreased at the second time point as compared with thefirst time point, the amount of decrease is at least about 2-fold, atleast about 5-fold, at least about 10-fold, at least about 20-fold, atleast about 30-fold, at least about 40-fold, at least about 50-fold, atleast about 60-fold, at least about 70-fold, at least about 80-fold, atleast about 90-fold, at least about 100-fold or more than at least about100-fold. In some embodiments, the amount of decrease is less than2-fold.

Although each of the methods disclosed herein have been described withrespect to dorsal and/or ventral PDX1-positive foregut endoderm cells,it will be appreciated that in certain embodiments, these methods can beused to produce compositions comprising the dorsal and/or ventralPDX1-positive foregut/midgut endoderm cells that are described hereinand/or the dorsal and/or ventral PDX1-positive endoderm cells of theposterior portion of the foregut that are described herein. Furthermore,any of the PDX1-positive endoderm cell types disclosed in thisspecification can be utilized in the screening methods described herein.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting.

EXAMPLES

Many of the examples below describe the use of pluripotent human cells.Methods of producing pluripotent human cells are well known in the artand have been described numerous scientific publications, including U.S.Pat. Nos. 5,453,357, 5,670,372, 5,690,926, 6,090,622, 6,200,806 and6,251,671 as well as U.S. Patent Application Publication No.2004/0229350, the disclosures of which are incorporated herein byreference in their entireties.

Example 1 Human ES Cells

For our studies of endoderm development we employed human embryonic stemcells, which are pluripotent and can divide seemingly indefinitely inculture while maintaining a normal karyotype. ES cells were derived fromthe 5-day-old embryo inner cell mass using either immunological ormechanical methods for isolation. In particular, the human embryonicstem cell line hESCyt-25 was derived from a supernumerary frozen embryofrom an in vitro fertilization cycle following informed consent by thepatient. Upon thawing the hatched blastocyst was plated on mouseembryonic fibroblasts (MEF), in ES medium (DMEM, 20% FBS, non essentialamino acids, beta-mercaptoethanol, ITS supplement). The embryo adheredto the culture dish and after approximately two weeks, regions ofundifferentiated hESCs were transferred to new dishes with MEFs.Transfer was accomplished with mechanical cutting and a brief digestionwith dispase, followed by mechanical removal of the cell clusters,washing and re-plating. Since derivation, hESCyt-25 has been seriallypassaged over 100 times. We employed the hESCyt-25 human embryonic stemcell line as our starting material for the production of definitiveendoderm.

It will be appreciated by those of skill in the art that stem cells orother pluripotent cells can also be used as starting material for thedifferentiation procedures described herein. For example, cells obtainedfrom embryonic gonadal ridges, which can be isolated by methods known inthe art, can be used as pluripotent cellular starting material.

Example 2 hESCyt-25 Characterization

The human embryonic stem cell line, hESCyt-25 has maintained a normalmorphology, karyotype, growth and self-renewal properties over 18 monthsin culture. This cell line displays strong immunoreactivity for theOCT4, SSEA-4 and TRA-1-60 antigens, all of which, are characteristic ofundifferentiated hESCs and displays alkaline phosphatase activity aswell as a morphology identical to other established hESC lines.Furthermore, the human stem cell line, hESCyt-25, also readily formsembryoid bodies (EBs) when cultured in suspension. As a demonstration ofits pluripotent nature, hESCyT-25 differentiates into various cell typesthat represent the three principal germ layers. Ectoderm production wasdemonstrated by Q-PCR for ZIC1 as well as immunocytochemistry (ICC) fornestin and more mature neuronal markers. Immunocytochemical staining forβ-III tubulin was observed in clusters of elongated cells,characteristic of early neurons. Previously, we treated EBs insuspension with retinoic acid, to induce differentiation of pluripotentstem cells to visceral endoderm (VE), an extra-embryonic lineage.Treated cells expressed high levels of α-fetoprotein (AFP) and SOX7, twomarkers of VE, by 54 hours of treatment. Cells differentiated inmonolayer expressed AFP in sporadic patches as demonstrated byimmunocytochemical staining. As will be described below, the hESCyT-25cell line was also capable of forming definitive endoderm, as validatedby real-time quantitative polymerase chain reaction (Q-PCR) andimmunocytochemistry for SOX17, in the absence of AFP expression. Todemonstrate differentiation to mesoderm, differentiating EBs wereanalyzed for Brachyury gene expression at several time points. Brachyuryexpression increased progressively over the course of the experiment. Inview of the foregoing, the hESCyT-25 line is pluripotent as shown by theability to form cells representing the three germ layers.

Example 3 Production of SOX17 Antibody

A primary obstacle to the identification of definitive endoderm in hESCcultures is the lack of appropriate tools. We therefore undertook theproduction of an antibody raised against human SOX17 protein.

The marker SOX17 is expressed throughout the definitive endoderm as itforms during gastrulation and its expression is maintained in the guttube (although levels of expression vary along the A-P axis) untilaround the onset of organogenesis. SOX17 is also expressed in a subsetof extra-embryonic endoderm cells. No expression of this protein hasbeen observed in mesoderm or ectoderm. It has now been discovered thatSOX17 is an appropriate marker for the definitive endoderm lineage whenused in conjunction with markers to exclude extra-embryonic lineages.

As described in detail herein, the SOX17 antibody was utilized tospecifically examine effects of various treatments and differentiationprocedures aimed at the production of SOX17 positive definitive endodermcells. Other antibodies reactive to AFP, SPARC and Thrombomodulin werealso employed to rule out the production of visceral and parietalendoderm (extra-embryonic endoderm).

In order to produce an antibody against SOX17, a portion of the humanSOX17 cDNA (SEQ ID NO: 1) corresponding to amino acids 172-414 (SEQ IDNO: 2) in the carboxyterminal end of the SOX17 protein (FIG. 2) was usedfor genetic immunization in rats at the antibody production company,GENOVAC (Freiberg, Germany), according to procedures developed there.Procedures for genetic immunization can be found in U.S. Pat. Nos.5,830,876, 5,817,637, 6,165,993 and 6,261,281 as well as InternationalPatent Application Publication Nos. WO00/29442 and WO99/13915, thedisclosures of which are incorporated herein by reference in theirentireties.

Other suitable methods for genetic immunization are also described inthe non-patent literature. For example, Barry et al. describe theproduction of monoclonal antibodies by genetic immunization inBiotechniques 16: 616-620, 1994, the disclosure of which is incorporatedherein by reference in its entirety. Specific examples of geneticimmunization methods to produce antibodies against specific proteins canbe found, for example, in Costaglia et al., (1998) Genetic immunizationagainst the human thyrotropin receptor causes thyroiditis and allowsproduction of monoclonal antibodies recognizing the native receptor, J.Immunol. 160: 1458-1465; Kilpatrick et al (1998) Gene gun deliveredDNA-based immunizations mediate rapid production of murine monoclonalantibodies to the Flt-3 receptor, Hybridoma 17: 569-576; Schmolke etal., (1998) Identification of hepatitis G virus particles in human serumby E2-specific monoclonal antibodies generated by DNA immunization, J.Virol. 72: 4541-4545; Krasemann et al., (1999) Generation of monoclonalantibodies against proteins with an unconventional nucleic acid-basedimmunization strategy, J. Biotechnol. 73: 119-129; and Ulivieri et al.,(1996) Generation of a monoclonal antibody to a defined portion of theHeliobacter pylori vacuolating cytotoxin by DNA immunization, J.Biotechnol. 51: 191-194, the disclosures of which are incorporatedherein by reference in their entireties.

SOX7 and SOX18 are the closest Sox family relatives to SOX17 as depictedin the relational dendrogram shown in FIG. 3. We employed the human SOX7polypeptide as a negative control to demonstrate that the SOX17 antibodyproduced by genetic immunization is specific for SOX17 and does notreact with its closest family member. In particular, SOX7 and otherproteins were expressed in human fibroblasts, and then, analyzed forcross reactivity with the SOX17 antibody by Western blot and ICC. Forexample, the following methods were utilized for the production of theSOX17, SOX7 and EGFP expression vectors, their transfection into humanfibroblasts and analysis by Western blot. Expression vectors employedfor the production of SOX17, SOX7, and EGFP were pCMV6 (OriGeneTechnologies, Inc., Rockville, Md.), pCMV-SPORT6 (Invitrogen, Carlsbad,Calif.) and pEGFP-N1 (Clonetech, Palo Alto, Calif.), respectively. Forprotein production, telomerase immortalized MDX human fibroblasts weretransiently transfected with supercoiled DNA in the presence ofLipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Total cellularlysates were collected 36 hours post-transfection in 50 mM TRIS-HCl (pH8), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, containing a cocktail ofprotease inhibitors (Roche Diagnostics Corporation, Indianapolis, Ind.).Western blot analysis of 100 μg of cellular proteins, separated bySDS-PAGE on NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad,Calif.), and transferred by electro-blotting onto PDVF membranes(Hercules, Calif.), were probed with a 1/1000 dilution of the rat SOX17anti-serum in 10 mM TRIS-HCl (pH 8), 150 mM NaCl, 10% BSA, 0.05%Tween-20 (Sigma, St. Louis, Mo.), followed by Alkaline Phosphataseconjugated anti-rat IgG (Jackson ImmunoResearch Laboratories, WestGrove, Pa.), and revealed through Vector Black Alkaline Phosphatasestaining (Vector Laboratories, Burlingame, Calif.). The proteins sizestandard used was wide range color markers (Sigma, St. Louis, Mo.).

In FIG. 4, protein extracts made from human fibroblast cells that weretransiently transfected with SOX17, SOX7 or EGFP cDNA's were probed onWestern blots with the SOX17 antibody. Only the protein extract fromhSOX17 transfected cells produced a band of ˜51 Kda which closelymatched the predicted 46 Kda molecular weight of the human SOX17protein. There was no reactivity of the SOX17 antibody to extracts madefrom either human SOX7 or EGFP transfected cells. Furthermore, the SOX17antibody clearly labeled the nuclei of human fibroblast cellstransfected with the hSOX17 expression construct but did not label cellstransfected with EGFP alone. As such, the SOX17 antibody exhibitsspecificity by ICC.

Example 4 Validation of SOX17 Antibody as a Marker of DefinitiveEndoderm

Partially differentiated hESCs were co-labeled with SOX17 and AFPantibodies to demonstrate that the SOX17 antibody is specific for humanSOX17 protein and furthermore marks definitive endoderm. It has beendemonstrated that SOX17, SOX7 (which is a closely related member of theSOX gene family subgroup F (FIG. 3)) and AFP are each expressed invisceral endoderm. However, AFP and SOX7 are not expressed in definitiveendoderm cells at levels detectable by ICC, and thus, they can beemployed as negative markers for bonifide definitive endoderm cells. Itwas shown that SOX17 antibody labels populations of cells that exist asdiscrete groupings of cells or are intermingled with AFP positive cells.In particular, FIG. 5A shows that small numbers of SOX17 cells wereco-labeled with AFP; however, regions were also found where there werelittle or no AFP cells in the field of SOX17⁺ cells (FIG. 5B).Similarly, since parietal endoderm has been reported to express SOX17,antibody co-labeling with SOX17 together with the parietal markers SPARCand/or Thrombomodulin (TM) can be used to identify the SOX17⁺ cells thatare parietal endoderm. As shown in FIGS. 6A-C, Thrombomodulin and SOX17co-labeled parietal endoderm cells were produced by randomdifferentiation of hES cells.

In view of the above cell labeling experiments, the identity of adefinitive endoderm cell can be established by the marker profileSOX17^(hi)/AFP^(lo)/[TM^(lo) or SPARC^(lo)]. In other words, theexpression of the SOX17 marker is greater than the expression of the AFPmarker, which is characteristic of visceral endoderm, and the TM orSPARC markers, which are characteristic of parietal endoderm.Accordingly, those cells positive for SOX17 but negative for AFP andnegative for TM or SPARC are definitive endoderm.

As a further evidence of the specificity of theSOX17^(hi)/AFP^(lo)/TM^(lo)/SPARC^(lo) marker profile as predictive ofdefinitive endoderm, SOX17 and AFP gene expression was quantitativelycompared to the relative number of antibody labeled cells. As shown inFIG. 7A, hESCs treated with retinoic acid (visceral endoderm inducer),or activin A (definitive endoderm inducer), resulted in a 10-folddifference in the level of SOX17 mRNA expression. This result mirroredthe 10-fold difference in SOX17 antibody-labeled cell number (FIG. 7B).Furthermore, as shown in FIG. 8A, activin A treatment of hESCssuppressed AFP gene expression by 6.8-fold in comparison to notreatment. This was visually reflected by a dramatic decrease in thenumber of AFP labeled cells in these cultures as shown in FIGS. 8B-C. Toquantify this further, it was demonstrated that this approximately7-fold decrease in AFP gene expression was the result of a similar7-fold decrease in AFP antibody-labeled cell number as measured by flowcytometry (FIGS. 9A-B). This result is extremely significant in that itindicates that quantitative changes in gene expression as seen by Q-PCRmirror changes in cell type specification as observed by antibodystaining.

Incubation of hESCs in the presence of Nodal family members (Nodal,activin A and activin B—NAA) resulted in a significant increase in SOX17antibody-labeled cells over time. By 5 days of continuous activintreatment greater than 50% of the cells were labeled with SOX17 (FIGS.10A-F). There were few or no cells labeled with AFP after 5 days ofactivin treatment.

In summary, the antibody produced against the carboxy-terminal 242 aminoacids of the human SOX17 protein identified human SOX17 protein onWestern blots but did not recognize SOX7, it's closest Sox familyrelative. The SOX17 antibody recognized a subset of cells indifferentiating hESC cultures that were primarily SOX17⁺/AFP^(lo/−)(greater than 95% of labeled cells) as well as a small percentage (<5%)of cells that co-label for SOX17 and AFP (visceral endoderm). Treatmentof hESC cultures with activins resulted in a marked elevation of SOX17gene expression as well as SOX17 labeled cells and dramaticallysuppressed the expression of AFP mRNA and the number of cells labeledwith AFP antibody.

Example 5 Q-PCR Gene Expression Assay

In the following experiments, real-time quantitative RT-PCR (Q-PCR) wasthe primary assay used for screening the effects of various treatmentson hESC differentiation. In particular, real-time measurements of geneexpression were analyzed for multiple marker genes at multiple timepoints by Q-PCR. Marker genes characteristic of the desired as well asundesired cell types were evaluated to gain a better understanding ofthe overall dynamics of the cellular populations. The strength of Q-PCRanalysis includes its extreme sensitivity and relative ease ofdeveloping the necessary markers, as the genome sequence is readilyavailable. Furthermore, the extremely high sensitivity of Q-PCR permitsdetection of gene expression from a relatively small number of cellswithin a much larger population. In addition, the ability to detect verylow levels of gene expression provides indications for “differentiationbias” within the population. The bias towards a particulardifferentiation pathway, prior to the overt differentiation of thosecellular phenotypes, is unrecognizable using immunocytochemicaltechniques. For this reason, Q-PCR provides a method of analysis that isat least complementary and potentially much superior toimmunocytochemical techniques for screening the success ofdifferentiation treatments. Additionally, Q-PCR provides a mechanism bywhich to evaluate the success of a differentiation protocol in aquantitative format at semi-high throughput scales of analysis.

The approach taken here was to perform relative quantitation using SYBRGreen chemistry on a Rotor Gene 3000 instrument (Corbett Research) and atwo-step RT-PCR format. Such an approach allowed for the banking of cDNAsamples for analysis of additional marker genes in the future, thusavoiding variability in the reverse transcription efficiency betweensamples.

Primers were designed to lie over exon-exon boundaries or span intronsof at least 800 by when possible, as this has been empiricallydetermined to eliminate amplification from contaminating genomic DNA.When marker genes were employed that do not contain introns or theypossess pseudogenes, DNase I treatment of RNA samples was performed.

We routinely used Q-PCR to measure the gene expression of multiplemarkers of target and non-target cell types in order to provide a broadprofile description of gene expression in cell samples. The markersrelevant for the early phases of hESC differentiation (specificallyectoderm, mesoderm, definitive endoderm and extra-embryonic endoderm)and for which validated primer sets are available are provided below inTable 1. The human specificity of these primer sets has also beendemonstrated. This is an important fact since the hESCs were often grownon mouse feeder layers. Most typically, triplicate samples were takenfor each condition and independently analyzed in duplicate to assess thebiological variability associated with each quantitative determination.

To generate PCR template, total RNA was isolated using RNeasy (Qiagen)and quantitated using RiboGreen (Molecular Probes). Reversetranscription from 350-500 ng of total RNA was carried out using theiScript reverse transcriptase kit (BioRad), which contains a mix ofoligo-dT and random primers. Each 20 μL reaction was subsequentlydiluted up to 100 μL total volume and 3 μL was used in each 10 μL Q-PCRreaction containing 400 nM forward and reverse primers and 5 μL 2×SYBRGreen master mix (Qiagen). Two step cycling parameters were usedemploying a 5 second denature at 85-94° C. (specifically selectedaccording to the melting temp of the amplicon for each primer set)followed by a 45 second anneal/extend at 60° C. Fluorescence data wascollected during the last 15 seconds of each extension phase. A threepoint, 10-fold dilution series was used to generate the standard curvefor each run and cycle thresholds (Ct's) were converted to quantitativevalues based on this standard curve. The quantitated values for eachsample were normalized to housekeeping gene performance and then averageand standard deviations were calculated for triplicate samples. At theconclusion of PCR cycling, a melt curve analysis was performed toascertain the specificity of the reaction. A single specific product wasindicated by a single peak at the T_(m) appropriate for that PCRamplicon. In addition, reactions performed without reverse transcriptaseserved as the negative control and do not amplify.

A first step in establishing the Q-PCR methodology was validation ofappropriate housekeeping genes (HGs) in the experimental system. Sincethe HG was used to normalize across samples for the RNA input, RNAintegrity and RT efficiency, it was of value that the HG exhibited aconstant level of expression over time in all sample types in order forthe normalization to be meaningful. We measured the expression levels ofCyclophilin G, hypoxanthine phosphoribosyltransferase 1 (HPRT),beta-2-microglobulin, hydroxymethylbiane synthase (HMBS), TATA-bindingprotein (TBP), and glucoronidase beta (GUS) in differentiating hESCs.Our results indicated that beta-2-microglobulin expression levelsincreased over the course of differentiation and therefore we excludedthe use of this gene for normalization. The other genes exhibitedconsistent expression levels over time as well as across treatments. Weroutinely used both Cyclophilin G and GUS to calculate a normalizationfactor for all samples. The use of multiple HGs simultaneously reducesthe variability inherent to the normalization process and increases thereliability of the relative gene expression values.

After obtaining genes for use in normalization, Q-PCR was then utilizedto determine the relative gene expression levels of many marker genesacross samples receiving different experimental treatments. The markergenes employed have been chosen because they exhibit enrichment inspecific populations representative of the early germ layers and inparticular have focused on sets of genes that are differentiallyexpressed in definitive endoderm and extra-embryonic endoderm. Thesegenes as well as their relative enrichment profiles are highlighted inTable 1.

TABLE 1 Germ Layer Gene Expression Domains Endoderm SOX17 definitive,visceral and parietal endoderm MIXL1 endoderm and mesoderm GATA4definitive and primitive endoderm HNF3b definitive endoderm andprimitive endoderm, mesoderm, neural plate Extra- GSC endoderm andmesoderm embryonic SOX7 visceral endoderm AFP visceral endoderm, liverSPARC parietal endoderm TM parietal endoderm/trophectoderm Ectoderm ZIC1neural tube, neural progenitors Mesoderm BRACH nascent mesoderm

Since many genes are expressed in more than one germ layer it is usefulto quantitatively compare expression levels of many genes within thesame experiment. SOX17 is expressed in definitive endoderm and to asmaller extent in visceral and parietal endoderm. SOX7 and AFP areexpressed in visceral endoderm at this early developmental time point.SPARC and TM are expressed in parietal endoderm and Brachyury isexpressed in early mesoderm.

Definitive endoderm cells were predicted to express high levels of SOX17mRNA and low levels of AFP and SOX7 (visceral endoderm), SPARC (parietalendoderm) and Brachyury (mesoderm). In addition, ZIC1 was used here tofurther rule out induction of early ectoderm. Finally, GATA4 and HNF3bwere expressed in both definitive and extra-embryonic endoderm, andthus, correlate with SOX17 expression in definitive endoderm (Table 1).A representative experiment is shown in FIGS. 11-14 which demonstrateshow the marker genes described in Table 1 correlate with each otheramong the various samples, thus highlighting specific patterns ofdifferentiation to definitive endoderm and extra-embryonic endoderm aswell as to mesodermal and neural cell types.

In view of the above data it is clear that increasing doses of activinresulted in increasing SOX17 gene expression. Further this SOX17expression predominantly represented definitive endoderm as opposed toextra-embryonic endoderm. This conclusion stems from the observationthat SOX17 gene expression was inversely correlated with AFP, SOX7, andSPARC gene expression.

Example 6 Directed Differentiation of Human ES Cells to DefinitiveEndoderm

Human ES cell cultures randomly differentiate if cultured underconditions that do not actively maintain their undifferentiated state.This heterogeneous differentiation results in production ofextra-embryonic endoderm cells comprised of both parietal and visceralendoderm (AFP, SPARC and SOX7 expression) as well as early ectodermaland mesodermal derivatives as marked by ZIC1 and Nestin (ectoderm) andBrachyury (mesoderm) expression. Definitive endoderm cell appearance hasnot been examined or specified for lack of specific antibody markers inES cell cultures. As such, and by default, early definitive endodermproduction in ES cell cultures has not been well studied. Sincesatisfactory antibody reagents for definitive endoderm cells have beenunavailable, most of the characterization has focused on ectoderm andextra-embryonic endoderm. Overall, there are significantly greaternumbers of extra-embryonic and neurectodermal cell types in comparisonto SOX17^(hi) definitive endoderm cells in randomly differentiated EScell cultures.

As undifferentiated hESC colonies expand on a bed of fibroblast feeders,the cells at the edges of the colony take on alternative morphologiesthat are distinct from those cells residing within the interior of thecolony. Many of these outer edge cells can be distinguished by theirless uniform, larger cell body morphology and by the expression ofhigher levels of OCT4. It has been described that as ES cells begin todifferentiate they alter the levels of OCT4 expression up or downrelative to undifferentiated ES cells. Alteration of OCT4 levels aboveor below the undifferentiated threshold may signify the initial stagesof differentiation away from the pluripotent state.

When undifferentiated colonies were examined by SOX17immunocytochemistry, occasionally small 10-15-cell clusters ofSOX17-positive cells were detected at random locations on the peripheryand at the junctions between undifferentiated hESC colonies. As notedabove, these scattered pockets of outer colony edges appeared to be someof the first cells to differentiate away from the classical ES cellmorphology as the colony expanded in size and became more crowded.Younger, smaller fully undifferentiated colonies (<1 mm; 4-5 days old)showed no SOX17 positive cells within or at the edges of the colonieswhile older, larger colonies (1-2 mm diameter, >5 days old) had sporadicisolated patches of SOX17 positive, AFP negative cells at the peripheryof some colonies or in regions interior to the edge that did not displaythe classical hESC morphology described previously. Given that this wasthe first development of an effective SOX17 antibody, definitiveendoderm cells generated in such early “undifferentiated” ES cellcultures have never been previously demonstrated.

Based on negative correlations of SOX17 and SPARC gene expression levelsby Q-PCR, the vast majority of these SOX17 positive, AFP negative cellswill be negative for parietal endoderm markers by antibody co-labeling.This was specifically demonstrated for TM-expressing parietal endodermcells as shown in FIGS. 15A-B. Exposure to Nodal factors activin A and Bresulted in a dramatic decrease in the intensity of TM expression andthe number of TM positive cells. By triple labeling using SOX17, AFP andTM antibodies on an activin treated culture, clusters of SOX17 positivecells that were also negative for AFP and TM were observed (FIGS.16A-D). These are the first cellular demonstrations of SOX17 positivedefinitive endoderm cells in differentiating hESC cultures (FIGS. 16A-Dand 17).

With the SOX17 antibody and Q-PCR tools described above we have exploreda number of procedures capable of efficiently programming hESCs tobecome SOX17^(hi)/AFP^(lo)/SPARC/TM^(lo) definitive endoderm cells. Weapplied a variety of differentiation protocols aimed at increasing thenumber and proliferative capacity of these cells as measured at thepopulation level by Q-PCR for SOX17 gene expression and at the level ofindividual cells by antibody labeling of SOX17 protein.

We were the first to analyze and describe the effect of TGFβ familygrowth factors, such as Nodal/activin/BMP, for use in creatingdefinitive endoderm cells from embryonic stem cells in in vitro cellcultures. In typical experiments, activin A, activin B, BMP orcombinations of these growth factors were added to cultures ofundifferentiated human stem cell line hESCyt-25 to begin thedifferentiation process.

As shown in FIG. 19, addition of activin A at 100 ng/ml resulted in a19-fold induction of SOX17 gene expression vs. undifferentiated hESCs byday 4 of differentiation. Adding activin B, a second member of theactivin family, together with activin A, resulted in a 37-fold inductionover undifferentiated hESCs by day 4 of combined activin treatment.Finally, adding a third member of the TGFβ family from the Nodal/Activinand BMP subgroups, BMP4, together with activin A and activin B,increased the fold induction to 57 times that of undifferentiated hESCs(FIG. 19). When SOX17 induction with activins and BMP was compared to nofactor medium controls 5-, 10-, and 15-fold inductions resulted at the4-day time point. By five days of triple treatment with activins A, Band BMP, SOX17 was induced more than 70 times higher than hESCs. Thesedata indicate that higher doses and longer treatment times of theNodal/activin TGFβ family members results in increased expression ofSOX17.

Nodal and related molecules activin A, B and BMP facilitate theexpression of SOX17 and definitive endoderm formation in vivo or invitro. Furthermore, addition of BMP results in an improved SOX17induction possibly through the further induction of Cripto, the Nodalco-receptor.

We have demonstrated that the combination of activins A and B togetherwith BMP4 result in additive increases in SOX17 induction and hencedefinitive endoderm formation. BMP4 addition for prolonged periods (>4days), in combination with activin A and B may induce SOX17 in parietaland visceral endoderm as well as definitive endoderm. In someembodiments of the present invention, it is therefore valuable to removeBMP4 from the treatment within 4 days of addition.

To determine the effect of TGFβ factor treatment at the individual celllevel, a time course of TGFβ factor addition was examined using SOX17antibody labeling. As previously shown in FIGS. 10A-F, there was adramatic increase in the relative number of SOX17 labeled cells overtime. The relative quantification (FIG. 20) shows more than a 20-foldincrease in SOX17-labeled cells. This result indicates that both thenumbers of cells as well SOX17 gene expression level are increasing withtime of TGFβ factor exposure. As shown in FIG. 21, after four days ofexposure to Nodal, activin A, activin B and BMP4, the level of SOX17induction reached 168-fold over undifferentiated hESCs. FIG. 22 showsthat the relative number of SOX17-positive cells was also doseresponsive. activin A doses of 100 ng/ml or more were capable ofpotently inducing SOX17 gene expression and cell number.

In addition to the TGFβ family members, the Wnt family of molecules mayplay a role in specification and/or maintenance of definitive endoderm.The use of Wnt molecules was also beneficial for the differentiation ofhESCs to definitive endoderm as indicated by the increased SOX17 geneexpression in samples that were treated with activins plus Wnt3a overthat of activins alone (FIG. 23).

All of the experiments described above were performed using a tissueculture medium containing 10% serum with added factors. Surprisingly, wediscovered that the concentration of serum had an effect on the level ofSOX17 expression in the presence of added activins as shown in FIGS.24A-C. When serum levels were reduced from 10% to 2%, SOX17 expressiontripled in the presence of activins A and B.

Finally, we demonstrated that activin induced SOX17⁺ cells divide inculture as depicted in FIGS. 25A-D. The arrows show cells labeled withSOX17/PCNA/DAPI that are in mitosis as evidenced by thePCNA/DAPI-labeled mitotic plate pattern and the phase contrast mitoticprofile.

Example 7 Chemokine Receptor 4 (CXCR4) Expression Correlates withMarkers for Definitive Endoderm and not Markers for Mesoderm, Ectodermor Visceral Endoderm

As described above, hESCs can be induced to differentiate to thedefinitive endoderm germ layer by the application of cytokines of theTGFβ family and more specifically of the activin/nodal subfamily.Additionally, we have shown that the proportion of fetal bovine serum(FBS) in the differentiation culture medium effects the efficiency ofdefinitive endoderm differentiation from hESCs. This effect is such thatat a given concentration of activin A in the medium, higher levels ofFBS will inhibit maximal differentiation to definitive endoderm. In theabsence of exogenous activin A, differentiation of hESCs to thedefinitive endoderm lineage is very inefficient and the FBSconcentration has much milder effects on the differentiation process ofhESCs.

In these experiments, hESCs were differentiated by growing in RPMImedium (Invitrogen, Carlsbad, Calif.; cat#61870-036) supplemented with0.5%, 2.0% or 10% FBS and either with or without 100 ng/ml activin A for6 days. In addition, a gradient of FBS ranging from 0.5% to 2.0% overthe first three days of differentiation was also used in conjunctionwith 100 ng/ml of activin A. After the 6 days, replicate samples werecollected from each culture condition and analyzed for relative geneexpression by real-time quantitative PCR. The remaining cells were fixedfor immunofluorescent detection of SOX17 protein.

The expression levels of CXCR4 varied dramatically across the 7 cultureconditions used (FIG. 26). In general, CXCR4 expression was high inactivin A treated cultures (A100) and low in those which did not receiveexogenous activin A (NF). In addition, among the A100 treated cultures,CXCR4 expression was highest when FBS concentration was lowest. Therewas a remarkable decrease in CXCR4 level in the 10% FBS condition suchthat the relative expression was more in line with the conditions thatdid not receive activin A (NF).

As described above, expression of the SOX17, GSC, MIXL1, and HNF3β genesis consistent with the characterization of a cell as definitiveendoderm. The relative expression of these four genes across the 7differentiation conditions mirrors that of CXCR4 (FIGS. 27A-D). Thisdemonstrates that CXCR4 is also a marker of definitive endoderm.

Ectoderm and mesoderm lineages can be distinguished from definitiveendoderm by their expression of various markers. Early mesodermexpresses the genes Brachyury and MOX1 while nascent neuro-ectodermexpresses SOX1 and ZIC1. FIGS. 28A-D demonstrate that the cultures whichdid not receive exogenous activin A were preferentially enriched formesoderm and ectoderm gene expression and that among the activin Atreated cultures, the 10% FBS condition also had increased levels ofmesoderm and ectoderm marker expression. These patterns of expressionwere inverse to that of CXCR4 and indicated that CXCR4 was not highlyexpressed in mesoderm or ectoderm derived from hESCs at thisdevelopmental time period.

Early during mammalian development, differentiation to extra-embryoniclineages also occurs. Of particular relevance here is thedifferentiation of visceral endoderm that shares the expression of manygenes in common with definitive endoderm, including SOX17. Todistinguish definitive endoderm from extra-embryonic visceral endodermone should examine a marker that is distinct between these two. SOX7represents a marker that is expressed in the visceral endoderm but notin the definitive endoderm lineage. Thus, culture conditions thatexhibit robust SOX17 gene expression in the absence of SOX7 expressionare likely to contain definitive and not visceral endoderm. It is shownin FIG. 28E that SOX7 was highly expressed in cultures that did notreceive activin A, SOX7 also exhibited increased expression even in thepresence of activin A when FBS was included at 10%. This pattern is theinverse of the CXCR4 expression pattern and suggests that CXCR4 is nothighly expressed in visceral endoderm.

The relative number of SOX17 immunoreactive (SOX17⁺) cells present ineach of the differentiation conditions mentioned above was alsodetermined. When hESCs were differentiated in the presence of high doseactivin A and low FBS concentration (0.5%-2.0%) SOX17⁺ cells wereubiquitously distributed throughout the culture. When high dose activinA was used but FBS was included at 10% (v/v), the SOX17⁺ cells appearedat much lower frequency and always appeared in isolated clusters ratherthan evenly distributed throughout the culture (FIGS. 29A and C as wellas B and E). A further decrease in SOX17⁺ cells was seen when noexogenous activin A was used. Under these conditions the SOX17⁺ cellsalso appeared in clusters and these clusters were smaller and much morerare than those found in the high activin A, low FBS treatment (FIGS. 29C and F). These results demonstrate that the CXCR4 expression patternsnot only correspond to definitive endoderm gene expression but also tothe number of definitive endoderm cells in each condition.

Example 8 Differentiation Conditions that Enrich for Definitive EndodermIncrease the Proportion of CXCR4 Positive Cells

The dose of activin A also effects the efficiency at which definitiveendoderm can be derived from hESCs. This example demonstrates thatincreasing the dose of activin A increases the proportion of CXCR4⁺cells in the culture.

hESCs were differentiated in RPMI media supplemented with 0.5%-2% FBS(increased from 0.5% to 1.0% to 2.0% over the first 3 days ofdifferentiation) and either 0, 10, or 100 ng/ml of activin A. After 7days of differentiation the cells were dissociated in PBS withoutCa²⁺/Mg²⁺ containing 2% FBS and 2 mM (EDTA) for 5 minutes at roomtemperature. The cells were filtered through 35 μm nylon filters,counted and pelleted. Pellets were resuspended in a small volume of 50%human serum/50% normal donkey serum and incubated for 2 minutes on iceto block non-specific antibody binding sites. To this, 1 μl of mouseanti-CXCR4 antibody (Abcam, cat#ab10403-100) was added per 50 μl(containing approximately 10⁵ cells) and labeling proceeded for 45minutes on ice. Cells were washed by adding 5 ml of PBS containing 2%human serum (buffer) and pelleted. A second wash with 5 ml of buffer wascompleted then cells were resuspended in 50 μl buffer per 10⁵ cells.Secondary antibody (FITC conjugated donkey anti-mouse; JacksonImmunoResearch, cat#715-096-151) was added at 5 μg/ml finalconcentration and allowed to label for 30 minutes followed by two washesin buffer as above. Cells were resuspended at 5×10⁶ cells/ml in bufferand analyzed and sorted using a FACS Vantage (Beckton Dickenson) by thestaff at the flow cytometry core facility (The Scripps ResearchInstitute). Cells were collected directly into RLT lysis buffer (Qiagen)for subsequent isolation of total RNA for gene expression analysis byreal-time quantitative PCR.

The number of CXCR4⁺ cells as determined by flow cytometry were observedto increase dramatically as the dose of activin A was increased in thedifferentiation culture media (FIGS. 30A-C). The CXCR4⁺ cells were thosefalling within the R4 gate and this gate was set using a secondaryantibody-only control for which 0.2% of events were located in the R4gate. The dramatically increased numbers of CXCR4⁺ cells correlates witha robust increase in definitive endoderm gene expression as activin Adose is increased (FIGS. 31A-D).

Example 9 Isolation of CXCR4 Positive Cells Enriches for DefinitiveEndoderm Gene Expression and Depletes Cells Expressing Markers ofMesoderm, Ectoderm and Visceral Endoderm

The CXCR4⁺ and CXCR4⁻ cells identified in Example 8 above were collectedand analyzed for relative gene expression and the gene expression of theparent populations was determined simultaneously.

The relative levels of CXCR4 gene expression was dramatically increasedwith increasing dose of activin A (FIG. 32). This correlated very wellwith the activin A dose-dependent increase of CXCR4⁺ cells (FIGS.30A-C). It is also clear that isolation of the CXCR4⁺ cells from eachpopulation accounted for nearly all of the CXCR4 gene expression in thatpopulation. This demonstrates the efficiency of the FACS method forcollecting these cells.

Gene expression analysis revealed that the CXCR4⁺ cells contain not onlythe majority of the CXCR4 gene expression, but they also contained geneexpression for other markers of definitive endoderm. As shown in FIGS.31A-D, the CXCR4⁺ cells were further enriched over the parent A100population for SOX17, GSC, HNF3B, and MIXL1. In addition, the CXCR4⁻fraction contained very little gene expression for these definitiveendoderm markers. Moreover, the CXCR4⁺ and CXCR4⁻ populations displayedthe inverse pattern of gene expression for markers of mesoderm, ectodermand extra-embryonic endoderm. FIGS. 33A-D shows that the CXCR4⁺ cellswere depleted for gene expression of Brachyury, MOX1, ZIC1, and SOX7relative to the A100 parent population. This A100 parent population wasalready low in expression of these markers relative to the low dose orno activin A conditions. These results show that the isolation of CXCR4⁺cells from hESCs differentiated in the presence of high activin A yieldsa population that is highly enriched for and substantially puredefinitive endoderm.

Example 10 Quantitation of Definitive Endoderm Cells in a CellPopulation Using CXCR4

To confirm the quantitation of the proportion of definitive endodermcells present in a cell culture or cell population as determinedpreviously herein and as determined in U.S. Provisional PatentApplication No. 60/532,004, entitled DEFINITIVE ENDODERM, filed Dec. 23,2003, the disclosure of which is incorporated herein by reference in itsentirety, cells expressing CXCR4 and other markers of definitiveendoderm were analyzed by FACS.

Using the methods such as those described in the above Examples, hESCswere differentiated to produce definitive endoderm. In particular, toincrease the yield and purity in differentiating cell cultures, theserum concentration of the medium was controlled as follows: 0.2% FBS onday 1, 1.0% FBS on day 2 and 2.0% FBS on days 3-6. Differentiatedcultures were sorted by FACS using three cell surface epitopes,E-Cadherin, CXCR4, and Thrombomodulin. Sorted cell populations were thenanalyzed by Q-PCR to determine relative expression levels of markers fordefinitive and extraembryonic-endoderm as well as other cell types.CXCR4 sorted cells taken from optimally differentiated cultures resultedin the isolation of definitive endoderm cells that were >98% pure.

Table 2 shows the results of a marker analysis for a definitive endodermculture that was differentiated from hESCs using the methods describedherein.

TABLE 2 Composition of Definitive Endoderm Cultures Percent PercentPercent Percent of Definitive Extraembryonic hES Marker(s) cultureEndoderm endoderm cells SOX17 70-80 100 Thrombomodulin <2 0 75 AFP <1 025 CXCR4 70-80 100 0 ECAD 10 0 100 other (ECAD neg.) 10-20 Total 100 100100 100

In particular, Table 2 indicates that CXCR4 and SOX17 positive cells(endoderm) comprised from 70%-80% of the cells in the cell culture. Ofthese SOX17-expressing cells, less than 2% expressed TM (parietalendoderm) and less than 1% expressed AFP (visceral endoderm). Aftersubtracting the proportion of TM-positive and AFP-positive cells(combined parietal and visceral endoderm; 3% total) from the proportionof SOX17/CXCR4 positive cells, it can be seen that about 67% to about77% of the cell culture was definitive endoderm. Approximately 10% ofthe cells were positive for E-Cadherin (ECAD), which is a marker forhESCs, and about 10-20% of the cells were of other cell types.

We have discovered that the purity of definitive endoderm in thedifferentiating cell cultures that are obtained prior to FACS separationcan be improved as compared to the above-described low serum procedureby maintaining the FBS concentration at ≦0.5% throughout the 5-6 daydifferentiation procedure. However, maintaining the cell culture at≦0.5% throughout the 5-6 day differentiation procedure also results in areduced number of total definitive endoderm cells that are produced.

Definitive endoderm cells produced by methods described herein have beenmaintained and expanded in culture in the presence of activin forgreater than 50 days without appreciable differentiation. In such cases,SOX17, CXCR4, MIXL1, GATA4, HNF313 expression is maintained over theculture period. Additionally, TM, SPARC, OCT4, AFP, SOX7, ZIC1 and BRACHwere not detected in these cultures. It is likely that such cells can bemaintained and expanded in culture for substantially longer than 50 dayswithout appreciable differentiation.

Example 11 Additional Marker of Definitive Endoderm Cells

In the following experiment, RNA was isolated from purified definitiveendoderm and human embryonic stem cell populations. Gene expression wasthen analyzed by gene chip analysis of the RNA from each purifiedpopulation. Q-PCR was also performed to further investigate thepotential of genes expressed in definitive endoderm, but not inembryonic stem cells, as a marker for definitive endoderm.

Human embryonic stem cells (hESCs) were maintained in DMEM/F12 mediasupplemented with 20% KnockOut Serum Replacement, 4 ng/ml recombinanthuman basic fibroblast growth factor (bFGF), 0.1 mM 2-mercaptoethanol,L-glutamine, non-essential amino acids and penicillin/streptomycin.hESCs were differentiated to definitive endoderm by culturing for 5 daysin RPMI media supplemented with 100 ng/ml of recombinant human activinA, fetal bovine serum (FBS), and penicillin/streptomycin. Theconcentration of FBS was varied each day as follows: 0.1% (first day),0.2% (second day), 2% (days 3-5).

Cells were isolated by fluorescence activated cell sorting (FACS) inorder to obtain purified populations of hESCs and definitive endodermfor gene expression analysis. Immuno-purification was achieved for hESCsusing SSEA4 antigen (R&D Systems, cat#FAB1435P) and for definitiveendoderm using CXCR4 (R&D Systems, cat#FAB170P). Cells were dissociatedusing trypsin/EDTA (Invitrogen, cat#25300-054), washed in phosphatebuffered saline (PBS) containing 2% human serum and resuspended in 100%human serum on ice for 10 minutes to block non-specific binding.Staining was carried out for 30 minutes on ice by adding 200 μl ofphycoerythrin-conjugated antibody to 5×10⁶ cells in 800 μl human serum.Cells were washed twice with 8 ml of PBS buffer and resuspended in 1 mlof the same. FACS isolation was carried out by the core facility of TheScripps Research Institute using a FACS Vantage (BD Biosciences). Cellswere collected directly into RLT lysis buffer and RNA was isolated byRNeasy according to the manufacturers instructions (Qiagen).

Purified RNA was submitted in duplicate to Expression Analysis (Durham,N.C.) for generation of the expression profile data using the Affymetrixplatform and U133 Plus 2.0 high-density oligonucleotide arrays. Datapresented is a group comparison that identifies genes differentiallyexpressed between the two populations, hESCs and definitive endoderm.Genes that exhibited a robust upward change in expression level overthat found in hESCs were selected as new candidate markers that arehighly characteristic of definitive endoderm. Select genes were assayedby Q-PCR, as described above, to verify the gene expression changesfound on the gene chip and also to investigate the expression pattern ofthese genes during a time course of hESC differentiation.

FIGS. 34A-M show the gene expression results for certain markers.Results are displayed for cell cultures analyzed 1, 3 and 5 days afterthe addition of 100 ng/ml activin A, CXCR4-expressing definitiveendoderm cells purified at the end of the five day differentiationprocedure (CXDE), and in purified hESCs. A comparison of FIGS. 34C andG-M demonstrates that the six marker genes, FGF17, VWF, CALCR, FOXQ1,CMKOR1 and CRIP1, exhibit an expression pattern that is almost identicalto each other and which is also identical to the pattern of expressionof CXCR4 and the ratio of SOX17/SOX7. As described previously, SOX17 isexpressed in both the definitive endoderm as well as in theSOX7-expressing extra-embryonic endoderm. Since SOX7 is not expressed inthe definitive endoderm, the ratio of SOX17/SOX7 provides a reliableestimate of definitive endoderm contribution to the SOX17 expressionwitnessed in the population as a whole. The similarity of panels G-L andM to panel C indicates that FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1are likely markers of definitive endoderm and that they are notsignificantly expressed in extra-embryonic endoderm cells.

It will be appreciated that the Q-PCR results described herein can befurther confirmed by ICC.

Example 12 Retinoic Acid and FGF-10 Induces PDX1 Specifically inDefinitive Endoderm Cultures

The following experiment demonstrates that RA and FGF-10 induces theexpression of PDX1 in definitive endoderm cells.

Human embryonic stem cells were cultured with or without activins forfour days. On day four, 1 μM RA and 50 ng/ml FGF-10 were added to thecell culture. Forty-eight hours after the RA/FGF-10 addition, theexpression of the PDX1 marker gene and other marker genes not specificto foregut endoderm were quantitated by Q-PCR.

The application of RA to definitive endoderm cells caused a robustincrease in PDX1 gene expression (see FIG. 35) without increasing theexpression of visceral endoderm (50×7, AFP), neural (SOX1, ZIC1), orneuronal (NFM) gene expression markers (see FIG. 36A-F). PDX1 geneexpression was induced to levels approximately 500-fold higher thanobserved in definitive endoderm after 48 hours exposure to 1 μM RA and50 ng/ml FGF-10. Furthermore, these results show that substantial PDX1induction occurred only in cell cultures which had been previouslydifferentiated to definitive endoderm (50×17) as indicated by the160-fold higher PDX1 expression found in the activin treated cellcultures relative to those cultures that received no activin prior to RAapplication.

Example 13 FGF-10 Provides Additional Increase in PDX1 Expression OverRA Alone

This Example shows that the combination of RA and FGF-10 induces PDX1expression to a greater extent than RA alone.

As in the previous Example, hESCs were cultured with or without activinsfor four days. On day four, the cells were treated with one of thefollowing: 1 μM RA alone; 1 μM RA in combination with either FGF-4 orFGF-10; or 1 μM RA in combination with both FGF-4 and FGF-10. Theexpression of PDX1, SOX7 and NFM were quantitated by Q-PCR ninety sixhours after RA or RA/FGF.

The treatment of hESC cultures with activin followed by retinoic acidinduced a 60-fold increase in PDX1 gene expression. The addition ofFGF-4 to the RA treatment induced slightly more PDX1 (approximately3-fold over RA alone). However, by adding FGF-10 and retinoic acidtogether, the induction of PDX1 was further enhanced 60-fold over RAalone (see FIG. 37A). This very robust PDX1 induction was greater than1400-fold higher than with no activin or RA/FGF treatment.Interestingly, addition of FGF-4 and FGF-10 simultaneously abolished thebeneficial effect of the FGF-10, producing only the modest PDX1 increaseattributed to FGF-4 addition.

Addition of RA/FGF-4 or RA/FGF-10 combinations did not increase theexpression of marker genes not associated with foregut endoderm whencompared to cells not exposed to RA/FGF combinations (see FIG. 37B-C).

Example 14 Retinoic Acid Dose Affects Anterior-Posterior (A-P) PositionIn Vitro

To determine whether the dose of RA affects A-P position in in vitrocell cultures, the following experiment was performed.

Human embryonic stem cells were cultured with or without activins forfour days. On day four, FGF-10 at 50 ng/ml was added to the culture incombination with RA at 0.04 μM, 0.2 μM or 1.0 μM. The expression of thePDX1 marker gene as well as other markers not specific for foregutendoderm were quantitated by Q-PCR.

The addition of retinoic acid at various doses, in combination withFGF-10 at 50 ng/ml, induced differential gene expression patterns thatcorrelate with specific anterior-posterior positional patterns. Thehighest dose of RA (1 μM) preferentially induced expression of anteriorendoderm marker (HOXA3) and also produced the most robust increase inPDX1 (FIG. 38A-B). The middle dose of RA (0.2 μM) induced midgutendoderm markers (CDX1, HOXC6) (see FIGS. 38C and 41E), while the lowestdose of RA (0.04 μM) preferentially induced a marker of hindgut endoderm(HOXA13) (see FIG. 38D). The RA dose had essentially no effect on therelative expression of either neural (SOX1) or neuronal (NFM) markers(see FIG. 38F-G). This example highlights the use of RA as a morphogenin vitro and in particular as a morphogen of endoderm derivatives ofdifferentiating hESCs.

Example 15 Use of B27 Supplement Enhances Expression of PDX1

PDX1 expression in definitive endoderm can be influenced by the use of anumber of factors and cell growth/differentiation conditions. In thefollowing experiment, we show that the use of B27 supplement enhancesthe expression of PDX1 in definitive endoderm cells.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hES cells grown on mouseembryonic fibroblast feeders with high dose activin A (100-200 ng/ml in0.5-2% FBS/DMEM/F12) for 4 days. The no activin A control received0.5-2% FBS/DMEM/F12 with no added activin A. At four days, culturesreceived either no activin A in 2% FBS (none), and in 2% serumreplacement (SR), or 50 ng/ml activin A together with 2 μM RA and 50ng/ml FGF-10 in 2% FBS/DMEM/F12 (none, +FBS, +B27) and similarly in 2%Serum replacement (SR). B27 supplement, (Gibco/BRL), was added as a 1/50dilution directly into 2% FBS/DMEM/F12 (+B27). Duplicate cell sampleswhere taken for each point, and total RNA was isolated and subjected toQ-PCR as previously described.

FIG. 39A-E shows that serum-free supplement B27 provided an additionalbenefit for induction of PDX1 gene expression without inducing anincrease in the expression of markers genes not specific for foregutendoderm as compared to such marker gene expression in cells grownwithout serum.

Example 16 Use of Activin B to Enhance Induction of PDX1

This Example shows that the use of activin B enhances thedifferentiation of PDX1-negative cells to PDX1-positive cells in invitro cell culture.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (50 ng/ml) in low serum/RPMIfor 6 days. The FBS dose was 0% on day one, 0.2% on day two and 2% ondays 3-6. The negative control for definitive endoderm production (NF)received 2% FBS/RPMI with no added activin A. In order to induce PDX1expression, each of the cultures received retinoic acid at 2 μM in 2%FBS/RPMI on day 6. The cultures treated with activin A on days onethrough five were provided with different dosing combinations of activinA and activin B or remained in activin A alone at 50 ng/ml. The noactivin A control culture (NF) was provided neither activin A noractivin B. This RA/activin treatment was carried out for 3 days at whichtime PDX1 gene expression was measured by Q-PCR from duplicate cellsamples.

FIG. 40A shows that the addition of activin B at doses ranging from10-50 ng/ml (a10, a25 and a50) in the presence of 25 ng/ml (A25) or 50ng/ml (A50) of activin A increased the PDX1 expression at least 2-foldover the culture that received only activin A at 50 ng/ml. The increasein PDX1 as a result of activin B addition was without increase in HNF6expression (see FIG. 40B), which is a marker for liver as well aspancreas at this time in development. This result suggests that theproportion of cells differentiating to pancreas had been increasedrelative to liver.

Example 17 Use of Serum Dose to Enhance Induction of PDX1

The expression of PDX1 in definitive endoderm cells is influenced by theamount of serum present in the cell culture throughout thedifferentiation process. The following experiment shows that the levelof serum in a culture during the differentiation of hESCs toPDX1-negative definitive endoderm has an effect on the expression ofPDX1 during further differentiation of these cells to PDX1-positiveendoderm.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (100 ng/ml) in lowserum/RPMI for 5 days. The FBS dose was 0.1% on day one, 0.5% on day twoand either 0.5%, 2% or 10% on days 3-5. The no activin A control (NF)received the same daily FBS/RPMI dosing, but with no added activin A.PDX1 expression was induced beginning at day 6 by the addition of RA.During days 6-7, cultures received retinoic acid at 2 μM in 0.5%FBS/RPMI, 1 μM on day 8 and 0.2 μM on day 9-11. The activin A waslowered to 50 ng/ml during retinoic acid treatment and was left absentfrom the no activin A control (NF).

FIG. 41A shows that the FBS dosing during the 3 day period of definitiveendoderm induction (days 3, 4 and 5) had a lasting ability to change theinduction of PDX1 gene expression during the retinoic acid treatment.This was without significant alteration in the expression pattern ofZIC1 (FIG. 41B) or SOX7 (FIG. 41C) gene expression.

Example 18 Use of Conditioned Medium to Enhance Induction of PDX1

Other factors and growth conditions which influence the expression ofPDX1 in definitive endoderm cells were also studied. The followingexperiment shows the effect of conditioned media on the differentiationof PDX1-negative definitive endoderm cells to PDX1-positive endodermcells.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (100 ng/ml) in lowserum/RPMI for 5 days. The FBS dose was 0.2% on day one, 0.5% on day twoand 2% on days 3-5.

The definitive endoderm cultures generated by 5 days of activin Atreatment were then induced to differentiate to PDX1 expressing endodermby the addition of RA in 2% FBS/RPMI containing activin A at 25 ng/mlfor four days. The RA was 2 μM for the first two days of addition, 1 μMon the third day and 0.5 μM on the fourth day. This base medium for PDX1induction was provided fresh (2A25R) or after conditioning for 24 hoursby one of four different cell populations. Conditioned media (CM) weregenerated from either mouse embryonic fibroblasts (MEFCM) or from hESCsthat were first differentiated for 5 days by one of three conditions; i)3% FBS/RPMI (CM2), or ii) activin A (CM3) or iii) bone morphogenicprotein 4 (BMP4) (CM4). Activin A or BMP4 factors were provided at 100ng/ml under the same FBS dosing regimen described above (0.2%, 0.5%,2%). These three different differentiation paradigms yield three verydifferent populations of human cells by which the PDX1 induction mediacan be conditioned. The 3% FBS without added growth factor (NF) yields aheterogeneous population composed in large part of extraembryonicendoderm, ectoderm and mesoderm cells. The activin A treated culture(A100) yields a large proportion of definitive endoderm and the BMP4treated culture (B100) yields primarily trophectoderm and someextraembryonic endoderm.

FIG. 42A shows that PDX1 was induced equivalently in fresh andconditioned media over the first two days of RA treatment. However, bythe third day PDX1 expression had started to decrease in fresh media andMEF conditioned media treatments. The differentiated hESCs producedconditioned media that resulted in maintenance or further increases inthe PDX1 gene expression at levels 3 to 4-fold greater than fresh media.The effect of maintaining high PDX1 expression in hESC-conditioned mediawas further amplified on day four of RA treatment achieving levels 6 to7-fold higher than in fresh media. FIG. 42B shows that the conditionedmedia treatments resulted in much lower levels of CDX1 gene expression,a gene not expressed in the region of PDX1 expressing endoderm. Thisindicates that the overall purity of PDX1-expressing endoderm was muchenhanced by treating definitive endoderm with conditioned mediagenerated from differentiated hESC cultures.

FIG. 43 shows that PDX1 gene expression exhibited a positive doseresponse to the amount of conditioned media applied to the definitiveendoderm cells. Total volume of media added to each plate was 5 ml andthe indicated volume (see FIG. 43) of conditioned media was diluted intofresh media (A25R). It is of note that just 1 ml of conditioned mediaadded into 4 ml of fresh media was still able to induce and maintainhigher PDX1 expression levels than 5 ml of fresh media alone. Thissuggests that the beneficial effect of conditioned media for inductionof PDX1 expressing endoderm is dependent on the release of somesubstance or substances from the cells into the conditioned media andthat this substance(s) dose dependently enhances production ofPDX1-expressing endoderm.

Example 19 Validation of Antibodies which Bind to PDX1

Antibodies that bind to PDX1 are useful tools for monitoring theinduction of PDX1 expression in a cell population. This Example showsthat rabbit polyclonal and IgY antibodies to PDX1 can be used to detectthe presence of this protein.

In a first experiment, IgY anti-PDX1 (IgY α-PDX1) antibody binding toPDX1 in cell lysates was validated by Western blot analysis. In thisanalysis, the binding of IgY α-PDX1 antibody to 50 μg of total celllysate from MDX12 human fibroblasts or MDX12 cells transfected 24 hrspreviously with a PDX1 expression vector was compared. The cell lysatesseparated by SDS-PAGE, transferred to a membrane by electroblotting, andthen probed with the IgY α-PDX1 primary antiserum followed by alkalinephosphatase conjugated rabbit anti-IgY (Rb α-IgY) secondary antibodies.Different dilutions of primary and secondary antibodies were applied toseparate strips of the membrane in the following combinations: A (500×dilution of primary, 10,000× dilution of secondary), B (2,000×,10,000×), C (500×, 40,000×), D (2,000×, 40,000), E (8,000×, 40,000×).

Binding was detected in cells transfected with the PDX1 expressionvector (PDX1-positive) at all of the tested antibody combinations.Binding was only observed in untransfected (PDX1-negative) fibroblastswhen using the highest concentrations of both primary and secondaryantibody together (combination A). Such non-specific binding wascharacterized by the detection of an additional band at a molecularweight slightly higher than PDX1 in both the transfected anduntransfected fibroblasts.

In a second experiment, the binding of polyclonal rabbit anti-PDX1 (Rbα-PDX1) antibody to PDX1 was tested by immunocytochemistry. To produce aPDX1 expressing cell for such experiments, MS1-V cells (ATCC # CRL-2460)were transiently transfected with an expression vector of PDX1-EGFP(constructed using pEGFP-N1, Clontech). Transfected cells were thenlabeled with Rb α-PDX1 and α-EGFP antisera. Transfected cells werevisualized by both EGFP fluorescence as well as α-EGFPimmunocytochemistry through the use of a Cy5 conjugated secondaryantibody. PDX1 immunofluorescence was visualized through the use of anα-Rb Cy3-conjugated secondary antibody.

Binding of the Rb α-PDX1 and the α-EGPF antibodies co-localized with GPFexpression.

Example 20 Immunocytochemistry of Human Pancreatic Tissue

This Example shows that antibodies having specificity for PDX1 can beused to identify human PDX1-positive cells by immunocytochemistry.

In a first experiment, paraffin embedded sections of human pancreas werestained for insulin with guinea pig anti-insulin (Gp α-Ins) primaryantibody at a 1/200 dilution followed by dog anti-guinea pig (D α-Gp)secondary antibody conjugated to Cy2 at a 1/100 dilution. In a secondexperiment, the same paraffin embedded sections of human pancreas werestained for PDX1 with IgY α-PDX1 primary antibody at a 1/4000 dilutionfollowed Rb α-IgY secondary antibody conjugated to AF555 at a 1/300dilution. The images collected from the first and second experimentswhere then merged. In a third experiment, cells that were stained withIgY α-PDX1 antibodies were also stained with DAPI.

Analysis of the human pancreatic sections revealed the presence ofstrong staining of islets of Langerhans. Although the strongest PDX1signal appeared in islets (insulin-positive), weak staining was alsoseen in acinar tissue (insulin-negative). DAPI and PDX1 co-stainingshows that PDX1 was mostly but not exclusively localized to the nucleus.

Example 21 Immunoprecipitation of PDX1 from Retinoic Acid Treated Cells

To further confirm PDX1 expression in definitive endoderm cells thathave been differentiated in the presence of RA and the lack of PDX1 indefinitive endoderm cells that have not been differentiated with RA, arabbit anti-PDX1 (Rb α-PDX1) antibody was used to immunoprecipitate PDX1from both RA differentiated and undifferentiated definitive endodermcells. Immunoprecipitated RA was detected by Western blot analysis usingIgY α-PDX1 antibody.

To obtain undifferentiated and differentiated definitive endoderm celllysates for immunoprecipitation, hESCs were treated for 5 days withactivin A at 100 ng/ml in low serum (definitive endoderm) followed bytreatment with activin A at 50 ng/ml and 2 μM all-trans RA for two days,1 μM for one day and 0.2 μM for one day (PDX1-positive foregutendoderm). As a positive control cell lysates were also prepared fromMS1-V cells (ATCC # CRL-2460) transfected with a PDX1 expression vector.PDX1 was immunoprecipitated by adding Rb α-PDX1 and rabbit-specificsecondary antibodies to each lysate. The precipitate was harvested bycentrifugation. Immunoprecipitates were dissolved in SDS-containingbuffer then loaded onto a polyacrylamide gel. After separation, theproteins were transferred to a membrane by electroblotting, and thenprobed with the IgY α-PDX1 primary antibody followed by labeled Rb α-IgYsecondary antibodies.

Immunoprecipitates collected from the MS1-V positive control cells aswell as those from day 8 (lane d8, three days after the start of RAtreatment) and day 9 (lane d9, four days after the start of RA) cellswere positive for PDX1 protein (FIG. 44). Precipitates obtained fromundifferentiated definitive endoderm cells (that is, day 5 cells treatedwith activin A—designated (A) in FIG. 44) and undifferentiated hESCs(that is, untreated day 5 cells—designated as (NF) in FIG. 44) werenegative for PDX1.

Example 22 Generation of PDX1 Promoter-EGFP Transgenic hESC Lines

In order to use the PDX1 marker for cell isolation, we geneticallytagged PDX1-positive foregut endoderm cells with an expressible reportergene. This Example describes the construction of a vector comprising areporter cassette which comprises a reporter gene under the control ofthe PDX1 regulatory region. This Example also describes the preparationof a cell, such as a human embryonic stem cell, transfected with thisvector as well as a cell having this reporter cassette integrated intoits genome.

PDX1-expressing definitive endoderm cell lines genetically tagged with areporter gene were constructed by placing a GFP reporter gene under thecontrol of the regulatory region (promoter) of the PDX1 gene. First, aplasmid construct in which EGFP expression is driven by the human PDX1gene promoter was generated by replacing the CMV promoter of vectorpEGFP-N1 (Clontech) with the human PDX1 control region (GenbankAccession No. AF192496, the disclosure of which is incorporated hereinby reference in its entirety), which comprises a nucleotide sequenceranging from about 4.4 kilobase pairs (kb) upstream to about 85 basepairs (bp) downstream of the PDX1 transcription start site. This regioncontains the characterized regulatory elements of the PDX1 gene, and itis sufficient to confer the normal PDX1 expression pattern in transgenicmice. In the resulting vector, expression of EFGP is driven by the PDX1promoter. In some experiments, this vector can be transfected intohESCs.

The PDX1 promoter/EGFP cassette was excised from the above vector, andthen subcloned into a selection vector containing the neomycinphosphotransferase gene under control of the phosphoglycerate kinase-1promoter. The selection cassette was flanked by flp recombinaserecognition sites to allow removal of the cassette. This selectionvector was linearized, and then introduced into hESCs using standardlipofection methods. Following 10-14 days of selection in G418,undifferentiated transgenic hESC clones were isolated and expanded.

Example 23 Isolation of PDX1-Positive Foregut Endoderm

The following Example demonstrates that hESCs comprising the PDX1promoter/EGFP cassette can be differentiated into PDX1-positive endodermcells and then subsequently isolated by fluorescence-activated cellsorting (FACS).

PDX1 promoter/EGFP transgenic hESCs were differentiated for 5 days inactivin A-containing media followed by two days in media comprisingactivin A and RA. The differentiated cells were then harvested bytrypsin digestion and sorted on a Becton Dickinson FACS Diva directlyinto RNA lysis buffer or PBS. A sample of single live cells was takenwithout gating for EGFP (Live) and single live cells were gated intoEGFP positive (GFP) and GFP negative (Neg) populations. In oneexperiment, the EGFP positive fraction was separated into two equallysized populations according to fluorescence intensity (Hi and Lo).

Following sorting, cell populations were analyzed by both Q-PCR andimmunocytochemistry. For Q-PCR analysis, RNA was prepared using QiagenRNeasy columns and then converted to cDNA. Q-PCR was conducted asdescribed previously. For immunocytochemistry analysis, cells weresorted into PBS, fixed for 10 minutes in 4% paraformaldehyde, andadhered to glass slides using a Cytospin centrifuge. Primary antibodiesto Cytokeratin19 (KRT19) were from Chemicon; to Hepatocyte nuclearfactor 3 beta (HNF3β) from Santa Cruz; to Glucose Transporter 2 (GLUT2)from R&D systems. Appropriate secondary antibodies conjugated to FITC(green) or Rhodamine (Red) were used to detect binding of the primaryantibodies.

A typical FACS sort of differentiated cells is shown in FIG. 45. Thepercent isolated PDX1-positive cells in this example was approximately7%, which varied depending on the differentiation efficiency from about1% to about 20%.

Sorted cells were further subjected to Q-PCR analysis. Differentiatedcells showed a correlation of EGFP fluorescence with endogenous PDX1gene expression. Compared to non-fluorescing cells, the EGFP positivecells showed a greater than 20-fold increase in PDX1 expression levels(FIG. 46). The separation of high and low EGFP intensity cells indicatedthat EGFP expression level correlated with PDX1 expression level (FIG.47). In addition to PDX1 marker analysis, sorted cells were subjected toQ-PCR analysis of several genes that are expressed in pancreaticendoderm. Products of each of these marker genes (NKX2.2, GLUT2, KRT19,HNF4α and HNF3β) were all enriched in the EGFP positive fraction (FIGS.48A-E). In contrast, the neural markers ZIC1 and GFAP were not enrichedin sorted EGFP expressing cells (FIGS. 49A and B).

By immunocytochemistry, virtually all the isolated PDX1-positive cellswere seen to express KRT19 and GLUT2. This result is expected for cellsof the pancreatic endoderm lineage. Many of these cells were also HNF3βpositive by antibody staining.

Example 24 Production of PDX1-Positive Dorsal and Ventral ForegutEndoderm

This Example describes the production of PDX1-positive, dorsally-biased,foregut endoderm as well as the production of PDX1-positive,ventrally-biased, foregut endoderm.

Definitive endoderm was produced from undifferentiated hESCs using athree or five day protocol in which activin A was provided to theculture medium at a concentration of 100 ng/ml each day. For both dorsaland ventral differentiation, the medium composition for the first fivedays was as follows: Day 1—RPMI+0% fetal bovine serum (FBS), Day2—RPMI+0.2% FBS, Day 3—RPMI+2.0% FBS, Day 4—RPMI+2.0% FBS and Day5—RPMI+2.0% FBS. For the ventral differentiation, definitive endodermwas produced for 3 days in activin A at 100 ng/ml and then exposed toBMP4 at 3 ng/ml and FGF10 at 50 ng/ml. BMP4/FGF10 addition was carriedout in RPMI+2% FBS for the first 2 days and then subsequently inConnaught Medical Research Labs (CMRL) medium (Invitrogen, Carlsbad,Calif.) (see, Parker R. C., et al. 1957. N.Y. Academy of Sciences 5:303,the disclosure of which is incorporated herein by reference in itsentirety) containing B27 supplement (1 part B27 to 200 parts medium byvolume—(1:200)) (Invitrogen, Carlsbad, Calif.). For the dorsaldifferentiation procedure, definitive endoderm was produced for 5 daysin activin A at 100 ng/ml and then exposed to retinoic acid (RA) at 2 μMand activin A at 25 ng/ml in CMRL medium containing B27 supplement(1:200).

In the RA-based, dorsal differentiation procedure, there was a stronginduction of PDX1 and a maintenance of HB9 expression with no inducedexpression of HHEX or albumin, which are ventral liver markers (FIGS.50A-D). In the ventral differentiation protocol, which does not use RAbut instead uses FGF10 and BMP, PDX1 gene expression was also stronglyinduced. In contrast to the RA treatment, HB9 (dorsal endoderm marker)expression was not maintained and ventral liver markers, such as albuminand HHEX, were strongly induced along with PDX1 (FIGS. 50A-D). Thesedata indicated that in the presence of RA, the foregut PDX1-expressingendoderm was devoid of liver (a ventral organ) markers and expresseddorsal markers like HB9. In the absence of RA, the PDX1 expression wasnot accompanied by high HB9 expression levels. Furthermore, theexpression of classical liver markers, such as albumin and HHEX,indicated that the definitive endoderm was preferentially executing aventral differentiation program since liver is exclusively derived fromthe ventral endoderm.

Example 25 Production of PDX1-Positive Ventral Foregut Endoderm Cells isDependent on Definitive Endoderm Formation

This Example describes the production of PDX1-positive,ventrally-biased, foregut endoderm from cultures comprising varyingamounts of definitive endoderm cells. Cultures with no definitiveendoderm show very little production of PDX1-positive, ventrally biased,foregut endoderm. As the initial amount of definitive endoderm cellsincrease, so does the production of ventrally-biased, foregut endoderm.

Four separate conditions were used to treat hESCs that result in varyingproportions of differentiation to definitive endoderm. All fourconditions utilized RPMI supplemented with 0% FBS on the first day, 0.2%FBS on second day, and 2% FBS on days 3 and 4. The four conditions wereas follows: (a) BMP4 at 100 ng/mL with SU5402 at 5 μM; (b) no exogenousgrowth factors; (c) activin A at 15 ng/mL; and (d) activin A at 100ng/ml. After the first four days of differentiation, the relative levelsof definitive endoderm produced were indicated by cerberus (CER) andSOX17 expression levels, whereby definitive endoderm was essentiallyabsent under condition (a), minimal under condition (b), present undercondition (c) and highly present under condition (d). All cultures werethen incubated for 2 days with BMP4 at 3 ng/mL, FGF10 at 50 ng/ml andKAAD-cyclopamine at 0.5 μM in a base medium of 2% FBS in RPMI followedby 6 days with BMP4 at 3 ng/mL, FGF10 at 50 ng/ml and KAAD-cyclopamineat 0.5 μM in a base medium composed of CMRL with 1:200 dilution of B27extract.

In the presence of SU5402 and BMP4, conditions under which no definitiveendoderm was produced as demonstrated by lack of CER and SOX17 geneexpression (FIGS. 51A and 51B), there was no induction of PDX1 oralbumin gene expression after treatment with BMP4/FGF10 (ventralendoderm condition) (FIGS. 51C and 51D). This was similarly true for theno growth factor condition (condition (b)), in which very minimal levelsof definitive endoderm were formed as indicated by the low levels of CERand SOX17 (FIGS. 51A and 51B). Although PDX1 and albumin gene expressionwas very low under the no growth factor condition (FIGS. 51C and 51D),the amount of gene expression was significantly greater than thatproduced from condition (a). The hESCs treated with intermediate (15ng/ml) and high (100 ng/ml) doses of activin A yielded robust definitiveendoderm differentiation, indicated by high SOX17 gene expression levels(FIG. 51B). The high dose activin treatment produced definitive endodermprimarily of anterior character as indicated by very high CER expressionlevels. Both the condition (c) and condition (d) treatments exhibitedrobust ventral endoderm differentiation as indicated by high level PDX1and albumin gene expression (FIGS. 51C and 51D). The levels of PDX1 andalbumin expression were greatest in the most anterior endoderm becauseanterior endoderm remains competent to differentiate to more posteriorendoderm fates while posterior endoderm cells have lost competence toacquire more anterior fates. These data strongly indicated that theproduction of ventral PDX1-expressing foregut endoderm and liver weredependent upon efficient production of definitive endoderm.

Example 26 BMP4 is not Necessary for PDX1-Positive Ventral ForegutEndoderm

This Example describes the production of PDX1-positive,ventrally-biased, foregut endoderm in the absence of BMP4.

Definitive endoderm was produced by exposing undifferentiated hESCs toactivin at 100 ng/mL in RPMI base medium supplemented with 0%, 0.2%, and2% FBS on days 1 through 3, respectively. After 3 days of activin Atreatment, the cultures were switched to a base medium composed of RPMIcontaining 2% FBS and maintained under one of the following conditions:(a) BMP4 at 3 ng/ml with FGF10 at 50 ng/ml and KAAD-cyclopamine at 0.5μM; (b) FGF10 at 50 ng/ml and KAAD-cyclopamine at 0.5 μM; or (c-e) noexogenous factors. After two days, the base medium was changed to CMRLplus B27 supplement (1:200) and cells were maintained according toconditions (a-c) above. Alternatively, cells were maintained with noexogenous factors in RPMI with B27 supplement (1:200) (condition (d)) orRPMI with 2% FBS (condition (e)). The same factor treatment conditionswere maintained for 8 more days of differentiation.

BMP4 was not needed to produce either PDX1-positive ventral foregut orliver endoderm cells as indicated by the robust induction of PDX1 andalbumin expression in the absence of BMP4 (FIGS. 52A and 52B). BMP4addition appeared to be less favorable for the production ofPDX1-positive ventral foregut endoderm but the addition of BMP4 to FGF10and KAAD-cyclopamine treatment does not decrease ventral foregut liverendoderm gene expression (FIGS. 52A and 52B). The use of CMRL with B27supplement had some ability to induce PDX1 expression in the absence ofadded factors (condition (c)) while RPMI with B27 (condition (d)) andRPMI with 2% FBS (condition (e)) did not exhibit any induction of PDX1expression (FIG. 52A). There did not appear to be a significant effectof base media on the induction of liver gene expression. In summary,FGF10 and KAAD-cyclopamine are sufficient to produce PDX1-positiveventral foregut endoderm.

Example 27 Markers for the Identification of PDX1-Positive Dorsal andVentral Foregut Endoderm

This Example describes markers useful for, among other things, theidentification, detection, enrichment, isolation, purification,targeting and/or validation of PDX1-positive dorsal and ventral foregutendoderm.

Cell cultures differentiated as described in Example 24 were subjectedto gene chip analysis to globally monitor the gene expression dynamicsoccurring during differentiation of hESC to definitive endoderm andfurther on to more mature dorsal and ventral endoderm phenotypes.Duplicate samples were isolated at the times indicated in Example 24.Gene expression profiles were determined using Affymetrix U133 plus 2.0high density oligonucleotide arrays by Expression Analysis (Durham,N.C.) according to their internal standard operating procedures. We haveevaluated the patterns of gene expression across these 7 conditions/timepoints through manual inspection as well as through hierarchicalclustering analyses. We have looked for patterns of gene expression thatmatch the temporal pattern of PDX1 expression (dorsal and ventral) tofind novel genes that are expressed in both ventral and dorsaldifferentiation paradigms.

Provided are genes that have a significant similarity in expressionpattern to PDX1, and thus, may be co-expressed in PDX1-expressingforegut endoderm cells The genes listed in Table 3 are expressed in boththe dorsal and ventral PDX1 differentiation. The genes in Table 4 aredorsally biased and are preferentially expressed in the dorsal PDX1pattern.

Table 3 lists 39 markers that are expressed in both dorsal and ventralPDX1-positive foregut endoderm. Column 1 provides the commonly knowngene symbol for each marker. Columns 2 through 4 provide the Unigene,LocusLink, and OMIM accession numbers, respectively. Column 5 describedthe Genebank accession number for a nucleic acid sequence which includesthe marker described in column 1. Finally, column 6 provides adescription of the functional activity of the polypeptide marker that isencoded by the listed genetic marker.

It will be appreciated that the accession numbers listed in Table 3 canbe used by those of ordinary skill in the art to retrieve specificinformation about each sequence described in the table, including boththe primary nucleic acid and polypeptide sequence of each of thesemarker.

TABLE 3 Markers expressed in both dorsal and ventral PDX1-positiveforegut endoderm Gene_Symbol Unigene LocusLink OMIM SeqDerivedFrom GeneDescriptor ANXA4 Hs.422986 307 106491 NM_001153 annexin A4 ASCL1Hs.524672 429 100790 BC001638 achaete-scute complex-like 1 (Drosophila)BNC1 Hs.459153 646 601930 NM_001717 basonuclin 1 C10orf30 Hs.498740222389 AW195407 Chromosome 10 open reading frame 30 C2orf23 Hs.36888465055 609139 BE535746 chromosome 2 open reading frame 23 C9orf150Hs.445356 286343 AI972386 chromosome 9 open reading frame 150 CDH6Hs.171054 1004 603007 BC000019 cadherin 6, type 2, K-cadherin (fetalkidney) DACH1 Hs.129452 1602 603803 AI650353 dachshund homolog 1(Drosophila) DUSP9 Hs.144879 1852 300134 NM_001395 dual specificityphosphatase 9 ELMOD1 Hs.495779 55531 AL359601 ELMO domain containing 1FLJ21462 fis Hs.24321 AW236803 CDNA clone IMAGE: 5273964, partial cdsFLJ22761 Hs.522988 80201 W81116 hypothetical protein FLJ22761 GABRA2Hs.116250 2555 137140 NM_000807 gamma-aminobutyric acid (GABA) Areceptor, alpha 2 GRIA3 Hs.377070 2892 305915 BC032004 glutamatereceptor, ionotrophic, AMPA 3 HNF4G Hs.241529 3174 605966 AI916600hepatocyte nuclear factor 4, gamma IDH2 Hs.513141 3418 147650 U52144isocitrate dehydrogenase 2 (NADP+), mitochondrial IL6R Hs.135087 3570147880 AV700030 interleukin 6 receptor KCNJ2 Hs.1547 3759 170390AF153820 potassium inwardly-rectifying channel, subfamily J, member 2KLF3 Hs.298658 51274 AA130132 Kruppel-like factor 3 (basic) LGALS3Hs.531081 3958 153619 AW085690 Lectin, galactoside-binding, soluble, 3(galectin 3) LGALS3 /// Hs.531081 3958/// 153619 BC001120 lectin,galactoside-binding, soluble, 3 (galectin 3) /// galectin-3 GALIGinternal gene LIPC Hs.188630 3990 151670 NM_000236 lipase, hepatic MEIS1Hs.526754 4211 601739 NM_002398 Meis1, myeloid ecotropic viralintegration site 1 homolog (mouse) NR2F1 Hs.519445 7025 132890 AI951185Nuclear receptor subfamily 2, group F, member 1 ONECUT2 Hs.194725 9480604894 NM_004852 one cut domain, family member 2 PAPPA Hs.494928 5069176385 AA148534 pregnancy-associated plasma protein A, pappalysin 1PDE3B Hs.445711 5140 602047 NM_000753 phosphodiesterase 3B,cGMP-inhibited PGPEP1 Hs.131776 54858 NM_017712 pyroglutamyl-peptidase IPMS2L1 Hs.520575 5379 605038 D38503 postmeiotic segregation increased2-like 1 SERPINF2 Hs.159509 5345 262850 NM_000934 serine (or cysteine)proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epitheliumderived factor), member 2 SLC27A2 Hs.11729 11001 603247 NM_003645 solutecarrier family 27 (fatty acid transporter), member 2 SLN Hs.334629 6588602203 NM_003063 sarcolipin SOX9 Hs.2316 6662 114290 NM_000346 SRY (sexdetermining region Y)-box 9 (campomelic dysplasia, autosomalsex-reversal) SULT2A1 Hs.515835 6822 125263 U08024 sulfotransferasefamily, cytosolic, 2A, dehydroepiandrosterone (DHEA)-preferring, member1 TFPI Hs.516578 7035 152310 BF511231 Tissue factor pathway inhibitor(lipoprotein-associated coagulation inhibitor) ZHX1 Hs.521264 11244604764 AI123518 zinc fingers and homeoboxes 1 ZNF467 Hs.112158 168544BE549732 zinc finger protein 467 ZNF503 Hs.195710 84858 AA603467 zincfinger protein 503 Hs.142869 AI935586 Transcribed locus

FIGS. 53A-E further illustrate the commonality of expression profilebetween PDX1 and markers selected from Table 3. In particular, FIGS.53A-E provide examples of genes that displayed nearly identical geneexpression patterns to that of PDX1 across the 7 conditions/timemonitored in this experiment. Pattern recognition to this degree ofsimilarity most likely reflects co-expression of these genes in the samecells that express PDX1, thus making these markers excellent novelcandidate markers for PDX1-positive foregut endoderm from both dorsaland ventral endoderm origins.

Table 4 lists 50 markers that are specifically and/or preferentiallyexpressed in dorsal PDX1-positive foregut endoderm. Column 1 providesthe commonly known gene symbol for each marker. Columns 2 through 4provide the Unigene, LocusLink, and OMIM accession numbers,respectively. Column 5 described the Genebank accession number for anucleic acid sequence which includes the marker described in column 1.Finally, column 6 provides a description of the functional activity ofthe polypeptide marker that is encoded by the listed genetic marker.

It will be appreciated that the accession numbers listed in Table 4 canbe used by those of ordinary skill in the art to retrieve specificinformation about each sequence described in the table, including boththe primary nucleic acid and polypeptide sequence of each of thesemarker.

TABLE 4 Markers expressed in dorsally-biased PDX1-positive foregutendoderm Gene_Symbol Unigene LocusLink OMIM SeqDerivedFrom GeneDescriptor ADORA2A Hs.197029 135 102776 NM_000675 adenosine A2a receptorAMSH-LP Hs.16229 57559 AI638611 associated molecule with the SH3 domainof STAM (AMSH) like protein BAIAP2L1 Hs.489237 55971 AA628400BAI1-associated protein 2-like 1 CD47 Hs.446414 961 601028 BG230614 CD47antigen (Rh-related antigen, integrin-associated signal transducer) CHN2Hs.203663 1124 602857 AK026415 Chimerin (chimaerin) 2 CLDN3 Hs.256401365 602910 BE791251 claudin 3 CPVL Hs.233389 54504 NM_031311carboxypeptidase, vitellogenic-like /// carboxypeptidase,vitellogenic-like CREB3L1 Hs.405961 90993 AF055009 cAMP responsiveelement binding protein 3-like 1 DACT1 Hs.48950 51339 607861 NM_016651dapper homolog 1, antagonist of beta-catenin (xenopus) DPP6 Hs.4906841804 126141 AW071705 Dipeptidylpeptidase 6 ELF3 Hs.67928 1999 602191AF017307 E74-like factor 3 (ets domain transcription factor,epithelial-specific) ENPP2 Hs.190977 5168 601060 L35594 ectonucleotidepyrophosphatase/phosphodiesterase 2 (autotaxin) EPB41L1 Hs.437422 2036602879 AA912711 erythrocyte membrane protein band 4.1-like 1 FAM46CHs.356216 54855 AL046017 family with sequence similarity 46, member CFAM49A Hs.467769 81553 NM_030797 family with sequence similarity 49,member A /// family with sequence similarity 49, member A FLJ30596Hs.81907 133686 AI453203 hypothetical protein FLJ30596 HOXA1 Hs.673973198 142955 S79910 homeo box A1 HOXA3 Hs.533357 3200 142954 AW137982homeo box A3 HOXB2 Hs.514289 3212 142967 NM_002145 homeo box B2 LAF4Hs.444414 3899 601464 AW085505 Lymphoid nuclear protein related to AF4LOC283658 Hs.87194 283658 AA233912 hypothetical protein LOC283658 MAFHs.134859 4094 177075 AF055376 v-maf musculoaponeurotic fibrosarcomaoncogene homolog (avian) MAG Hs.515354 4099 159460 X98405 myelinassociated glycoprotein MYCPBP Hs.513817 10260 600382 BE268538 c-mycpromoter binding protein NR4A2 Hs.165258 4929 168600/ NM_006186 nuclearreceptor subfamily 4, group A, member 2 NRXN3 Hs.368307 9369 600567AI129949 neurexin 3 NSE1 Hs.260855 151354 AI601101 NSE1 PCGF5 Hs.50051284333 AL045882 polycomb group ring finger 5 PDE11A Hs.130312 50940604961 AB038041 phosphodiesterase 11A PDE5A Hs.370661 8654 603310BF221547 Phosphodiesterase 5A, cGMP-specific PGA3 5220 169710 AI570199pepsinogen 3, group I (pepsinogen A) PLN Hs.170839 5350 115200 NM_002667phospholamban PTGIS Hs.302085 5740 145500 NM_000961 prostaglandin I2(prostacyclin) synthase /// prostaglandin I2 (prostacyclin) synthaseRARB Hs.436538 5915 180220 NM_000965 retinoic acid receptor, beta RGNHs.77854 9104 300212 D31815 regucalcin (senescence marker protein-30)RND1 Hs.124940 27289 609038 U69563 Rho family GTPase 1 SFRP5 Hs.2795656425 604158 NM_003015 secreted frizzled-related protein 5 SGKL Hs.38087723678 607591 AV690866 serum/glucocorticoid regulated kinase-likeSLC16A10 Hs.520321 117247 607550 N30257 solute carrier family 16(monocarboxylic acid transporters), member 10 SLC16A2 Hs.75317 6567300095 NM_006517 solute carrier family 16 (monocarboxylic acidtransporters), member 2 SLC1A3 Hs.481918 6507 600111 NM_004172 solutecarrier family 1 (glial high affinity glutamate transporter), member 3SLC30A4 Hs.162989 7782 602095 NM_013309 solute carrier family 30 (zinctransporter), member 4 SLICK Hs.420016 343450 AI732637 sodium- andchloride-activated ATP-sensitive potassium channel SLITRK4 Hs.272284139065 AL080239 SLIT and NTRK-like family, member 4 ST8SIA3 Hs.29892351046 NM_015879 ST8 alpha-N-acetyl-neuraminidealpha-2,8-sialyltransferase 3 WNT5A Hs.152213 7474 164975 AI968085wingless-type MMTV integration site family, member 5A /// wingless-typeMMTV integration site family, member 5A XPR1 Hs.227656 9213 605237AF089744 xenotropic and polytropic retrovirus receptor Hs.535688AK001582 CDNA FLJ10720 fis, clone NT2RP3001116 Hs.127009 AI935541Transcribed locus Hs.4749 AL137310 CDNA FLJ31660 fis, clone NT2RI2004410

FIG. 54A-D provide examples of genes that display patterns of geneexpression that indicate specific (HOXA1 and PDE11A) or preferential(FAM49A and WNT5A) expression in the dorsal endoderm condition (RAtreatment). These markers are novel candidate genes for identificationof PDX1-positive, dorsally-biased, foregut endoderm.

Example 28 Production of PDX1-Negative Foregut Endoderm

This Example describes the production of PDX1-negative foregut endoderm.

Human embryonic stem cells were differentiated for 7 days via a 2-stepprotocol to achieve PDX1 cells. The first step comprised 5 daysdifferentiation in activin A (100 ng/ml) to robustly produce DE (D′Amour, K., et al., Nature Biotechnology 23, 1534-1541, (2005)). Step 2comprised 2 days differentiation in fresh RPMI with 2% FBS containingFGF10 (50 ng/ml) and KAAD-cyclopamine (0.5 μM).

The addition of FGF10 (5-500 ng/ml) was beneficial together with theaddition of KAAD-cyclopamine (0.1-2 μM, sonic hedgehog inhibitor), whichfurther specified definitive endoderm cells into the foregut endodermdomain.

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

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Numerous literature and patent references have been cited in the presentpatent application. Each and every reference that is cited in thispatent application is incorporated by reference herein in its entirety.

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1. A cell culture comprising human cells wherein at least 10% of saidhuman cells are pancreatic-duodenal homoebox factor-1 (PDX1) positive,endoderm cells that express at least one marker selected from the groupconsisting of SOX9, HNF6, and PROX1, and which express the PDX1 protein.2. The cell culture of claim 1, wherein at least 40% of said human cellsare PDX1-positive endoderm cells.
 3. The cell culture of claim 1,wherein at least 50% of said human cells are PDX1-positive endodermcells.
 4. The cell culture of claim 1, wherein at least 60% of saidhuman cells are PDX1-positive endoderm cells.
 5. The cell culture ofclaim 1, wherein at least 75% of said human cells are PDX1-positiveendoderm cells.
 6. The cell culture of claim 1, wherein human feedercells are present in said culture, and wherein at least 10% of humancells other than said human feeder cells are PDX1-positive endodermcells.
 7. The cell culture of claim 1, wherein the expression of PDX1 isgreater than the expression of a marker selected from the groupconsisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in saidPDX1-positive endoderm cells, wherein expression is determined byquantitative PCR.
 8. The cell culture of claim 1, wherein said cellculture is substantially free of cells selected from the groupconsisting of visceral endodermal cells, parietal endodermal cells andneural cells.
 9. The cell culture of claim 1 further comprising aretinoid.
 10. The cell culture of claim 9, wherein said retinoid isretinoic acid (RA).
 11. The cell culture of claim 10 further comprisingB27.
 12. The cell culture of claim 1, wherein said human cells arenon-recombinant.
 13. The cell culture of claim 1, wherein said endodermcells are multipotent cells that can further differentiate to cellsderived from the foregut.
 14. A cell population comprising cells whereinat least 90% of said cells are human PDX1-positive, endoderm cells thatexpress at least one marker selected from the group consisting of SOX9,HNF6, and PROX1, and which express the PDX1 protein.
 15. The cellpopulation of claim 14, wherein at least 98% of said cells arePDX1-positive endoderm cells.
 16. The cell population of claim 14,wherein the expression of PDX1 is greater than the expression of amarker selected from the group consisting of AFP, SOX7, SOX1, ZIC1 andNFM in said PDX1-positive endoderm cells, wherein expression isdetermined by quantitative PCR.
 17. The cell population of claim 14,wherein said human cells are non-recombinant.
 18. The cell population ofclaim 14, wherein said endoderm cells are multipotent cells that canfurther differentiate to cells derived from the foregut.
 19. A method ofproducing PDX1-positive endoderm cells, said method comprising: (a)culturing a population of pluripotent human cells in the presence ofexogenous TGFβ superfamily growth factor; (b) culturing the cells fromstep (a) in the presence of an exogenous FGF-family growth factor; and(c) culturing the cells from step (b) in the presence of a retinoid,thereby producing multipotent PDX1-positive endoderm cells.
 20. Themethod of claim 19, wherein said TGFβ superfamily growth factorcomprises activin A.
 21. The method of claim 19, wherein the FGF-familygrowth factor comprises FGF-7.
 22. The method of claim 19, wherein saidretinoid is retinoic acid (RA).
 23. The method of claim 22, wherein RAis provided in a concentration ranging from 0.01 μM to about 50 μM. 24.The method of claim 19, wherein at least 25% of said cell populationexpresses PDX1.
 25. A method of screening growth factors capable ofpromoting the differentiation of PDX1-negative definitive endoderm cellsto PDX1-positive definitive endoderm cells, said method comprising: (a)culturing a population of PDX1-negative definitive endoderm cells; (b)determining the expression of PDX1 in said population; (c) providing oneor more candidate growth factors to said population; (d) determining theexpression of PDX1 in said population after addition of the one or morecandidate growth factors; (e) comparing expression of PDX1 at step (b)with expression of PDX1 at step (d); (f) evaluating whether expressionof PDX1 at step (d) is increased as compared to expression of PDX1 atstep (b) thereby determining that the candidate differentiation factoris capable of promoting the differentiation of PDX1-negative definitiveendoderm cells to PDX1-positive definitive endoderm cells.